Inhibition of complement activation

ABSTRACT

The invention relates to factor D inhibitors, which bind to factor D and block the functional activity of factor D in complement activation. The inhibitors include antibody molecules, as well as homologues, analogues and modified or derived forms thereof, including immunoglobulin fragments like Fab, F(ab′) 2  and Fv, small molecules, including peptides, oligonucleotides, peptidomimetics and organic compounds. A monoclonal antibody which bound to factor D and blocked its ability to activate complement was generated and designated 166-32. The hybridoma producing this antibody was deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, under Accession Number HB-12476.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/139,447, which is a divisional of U.S. application Ser. No.09/821,255, which is a continuation-in-part of U.S. application Ser. No.09/253,689, filed on Feb. 20, 1999, which claims priority to U.S.Provisional Application No. 60/075,328, filed on Feb. 20, 1998.

FIELD OF THE INVENTION

This invention relates to inhibitors specific to factor D and the use ofsuch inhibitors in inhibiting complement system activation and, moreparticularly, in inhibiting alternative pathway complement activation.

BACKGROUND OF THE INVENTION

The complement system plays a central role in the clearance of immunecomplexes and the immune response to infectious agents, foreignantigens, virus-infected cells and tumor cells. However, complement isalso involved in pathological inflammation and in autoimmune diseases.Therefore, inhibition of excessive or uncontrolled activation of thecomplement cascade could provide clinical benefit to patients with suchdiseases and conditions.

The complement system encompasses two distinct activation pathways,designated the classical and the alternative pathways (V. M. Holers, InClinical Immunology: Principles and Practice, ed. R. R. Rich, MosbyPress; 1996, 363-391). The classical pathway is acalcium/magnesium-dependent cascade which is normally activated by theformation of antigen-antibody complexes. The alternative pathway is amagnesium-dependent cascade which is activated by deposition andactivation of C3 on certain susceptible surfaces (e.g. cell wallpolysaccharides of yeast and bacteria, and certain biopolymermaterials). Activation of the complement pathway generates biologicallyactive fragments of complement proteins, e.g. C3a, C4a and C5aanaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediateinflammatory activities involving leukocyte chemotaxis, activation ofmacrophages, neutrophils, platelets, mast cells and endothelial cells,vascular permeability, cytolysis, and tissue injury.

Factor D is a highly specific serine protease essential for activationof the alternative complement pathway. It cleaves factor B bound to C3b,generating the C3b/Bb enzyme which is the active component of thealternative pathway C3/C5 convertases. Factor D may be a suitable targetfor inhibition, since its plasma concentration in humans is very low(1.8 μg/ml), and it has been shown to be the limiting enzyme foractivation of the alternative complement pathway (P. H. Lesavre and H.J. Müller-Eberhard. J. Exp. Med., 1978; 148: 1498-1510; J. E. Volanakiset al., New Eng. J. Med., 1985; 312: 395-401).

The down-regulation of complement activation has been demonstrated to beeffective in treating several disease indications in animal models andin ex vivo studies, e.g. systemic lupus erythematosus andglomerulonephritis (Y. Wang et al., Proc. Natl. Acad. Sci.; 1996, 93:8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad.Sci., 1995; 92: 8955-8959), cardiopulmonary bypass and hemodialysis (C.S. Rinder, J. Clin. Invest., 1995; 96: 1564-1572), hypercute rejectionin organ transplantation (T. J. Kroshus et al., Transplantation, 1995;60: 1194-1202), myocardial infarction (J. W. Homeister et al., J.Immunol., 1993; 150: 1055-1064; H. F. Weisman et al., Science, 1990;249: 146-151), reperfusion injury (E. A. Amsterdam et al., Am. J.Physiol., 1995; 268: H448-H457), and adult respiratory distress syndrome(R. Rabinovici et al., J. Immunol., 1992; 149: 1744-1750). In addition,other inflammatory conditions and autoimmune/immune complex diseases arealso closely associated with complement activation (V. M. Holers, ibid.,B. P. Morgan. Eur. J. Clin. Invest., 1994:24:219-228), including thermalinjury, severe asthma, anaphylactic shock, bowel inflammation,urticaria, angioedema, vasculitis, multiple sclerosis, myastheniagravis, membranoproliferative glomerulonephritis, and Sjögren'ssyndrome.

SUMMARY OF THE INVENTION

The invention includes factor D inhibitors, which bind to factor D andblock the functional activity of factor D complement activation,including in alternative pathway complement activation. The inhibitorsinclude antibody molecules, as well as homologues, analogues andmodified or derived forms thereof, including immunoglobulin fragmentslike Fab, F(ab′)₂ and Fv. Small molecules including peptides,oligonucleotides, peptidomimetics and organic compounds which bind tofactor D and block its functional activity are also included.

A monoclonal antibody which bound to factor D and blocked its ability toactivate complement was generated and designated 166-32. The hybridomaproducing this antibody was deposited at the American Type CultureCollection, 10801 University Blvd., Manassas, Va. 20110-2209, underAccession Number HB-12476.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of anti-factor D monoclonal antibodies (MAbs)to purified human factor D in ELISA. The line marked with filled circlesrepresents MAb 166-11. The line marked with filled triangles representsMAb 166-32. The line marked with filled diamonds represents MAb 166-188.The line marked with filled squares represents MAb 166-222. The Y-axisrepresents the reactivity of the MAbs with factor D expressed as opticaldensity (OD) at 450 nm and the X-axis represents the concentration ofthe MAbs.

FIG. 2 shows the inhibition of alternative pathway (AP) hemolysis ofunsensitized rabbit red blood cells (RBCs) by MAb 166-32 in the presenceof 10% human serum. The line marked with filled squares represents MAb166-32. The line marked with filled circles represents the irrelevantisotype-matched control MAb G3-519, which is specific to HIV envelopeglycoprotein gp120. The Y-axis represents the % hemolysis inhibition, asfurther described in the text. The X-axis represents the concentrationof the MAbs.

FIG. 3 shows the inhibition of alternative pathway (AP) hemolysis ofunsensitized rabbit red blood cells (RBCs) by MAb 166-32 in the presenceof 90% human serum. The line marked with filled squares represents MAb166-32. The line marked with filled circles represents the irrelevantisotype-matched control MAb G3-519, which is specific to HIV envelopeglycoprotein gp120. The Y-axis represents the % hemolysis inhibition, asfurther described in the text. The X-axis represents the concentrationof the MAbs.

FIG. 4 shows that MAb 166-32 does not inhibit classical pathway (CP)hemolysis of sensitized chicken RBCs, whereas the positive controlanti-human C5 MAb 137-76 does. The line marked with filled circlesrepresents MAb 137-76. The line marked with filled diamonds and withfilled squares represents MAb 166-32 and the negative control MAbG3-519, respectively. The Y-axis represents the % hemolysis inhibition.The X-axis represents the concentration of the MAbs.

FIG. 5 shows the inhibition of alternative pathway (AP) hemolysis by MAb166-32. Hemolysis was augmented by adding different concentrations ofpurified human factor D to a human serum depleted of its factor D byaffinity chromatography using anti-factor D MAb 166-222. The assays wereperformed in the presence or absence of 0.3 μg/ml test MAbs. The linemarked with filled squares represents no antibody added. The line markedwith filled circles represents MAb 166-32. The line marked with filledtriangles represents the irrelevant isotype-matched control MAb G3-519.The Y-axis represents the % hemolysis inhibition. The X-axis representsthe concentration of factor D.

FIG. 6 shows the inhibition of factor-D dependent EAC3b cell lysis byMAb 166-32. The alternative C3 convertase was assembled on EAC3b cellsby incubation with factor B, factor P and factor D. Differentconcentrations of MAb 166-32 were added to the incubation buffer toinhibit the activity of factor D. The line marked with filled squaresrepresents MAb 166-32. The line marked with filled circles representsMAb G3-519. The Y-axis represents the % hemolysis inhibition. The X-axisrepresents the concentration of the MAbs.

FIG. 7 shows the inhibition of C3a production from zymosan by MAb166-32. Zymosan activated the alternative complement pathway in thepresence of human serum. The production of C3a was measured by using anELISA assay kit. The line marked with filled squares represents MAb166-32. The line marked with filled circles represents the irrelevantisotype-control MAb G3-519. The Y-axis represents the % inhibition ofC3a production. The X-axis represents the concentration of the MAbs.

FIG. 8 shows the inhibition of sC5b-9 production from zymosan by MAb166-32. Zymosan activated the alternative complement pathway in thepresence of human serum. The production of sC5b-9 was measured by usingan ELISA assay kit. The line marked with filled squares represents MAb166-32. The line marked with filled circles represents the irrelevantisotype-control MAb G3-519. The Y-axis represents the % inhibition ofsC5b-9 production. The X-axis represents the concentration of the MAbs.

FIG. 9 shows the inhibition of alternative pathway hemolysis ofunsensitized rabbit RBCs by MAb 166-32 and its Fab. The line marked withfilled circles represents MAb 166-32 (whole IgG). The line marked withfilled squares represents the Fab of the MAb 166-32. The Y-axisrepresents the % hemolysis inhibition. The X-axis represents theconcentration of the MAbs.

FIG. 10 shows the inhibitory effect of MAb 166-32 on factor D in serafrom different animal species in alternative pathway hemolysis ofunsensitized rabbit RBCs. The line marked with filled squares representshuman serum. The line marked with filled circles represents chimpanzeeserum. The line marked with filled triangles represents rhesus monkeyserum. The line marked with filled, inverted triangles represents baboonserum. The line marked with filled diamonds represents cynomolgus monkeyserum. The line marked with open circles represents sheep serum. Theline marked with open triangles represents canine serum. The Y-axisrepresents the % hemolysis inhibition. The X-axis represents theconcentration of MAb 166-32.

FIG. 11 shows the reactivity of MAb 166-32 with different Baculovirusexpressed factor D (“FD”) mutants and hybrids in ELISA. The line markedwith filled squares represents human factor D, FD/Hu. The line markedwith filled circles represents pig factor D, FD/Pig. The line markedwith filled triangles represents FD/Pighu. The line marked with filled,inverted triangles represents the hybrid protein, FD/Hupig. The linemarked with filled diamonds represents the mutant protein, FD/VDA. Theline marked with open circles represents the mutant protein, FD/L. Theline marked with open triangles represents the mutant protein, FD/RH.The line marked with open diamonds represents the blank with no coatingantigen. The recombinant proteins are further described in the text.

FIG. 12 shows the schematic representation of the expression vectorplasmids for chimeric 166-32 Fab: (A) pSV2dhfrFd and (B) pSV2neok. Solidboxes represent the exons encoding the Fd or κ gene. Hatched segmentsrepresent the HCMV-derived enhancer and promoter elements (E-P), asindicated below. Open boxes are the dihydrofolate reductase (dhfr) andneo genes, as marked. The pSV2 plasmid consists of DNA segments fromvarious sources: pBR322DNA (thin line) contains the pBR322 origin of DNAreplication (pBR ori) and the lactamase ampicillin resistance gene(Amp); SV40 DNA, represented by wider hatching and marked, contains theSV40 origin of DNA replication (SV40 ori), early promoter (5′ to thedhfr and neo genes), and polyadenylation signal (3′ to the dhfr and neogenes). The SV40-derived polyadenylation signal (pA) is also placed atthe 3′ end of the Fd gene.

FIG. 13 shows the inhibition of alternative pathway (AP) hemolysis ofunsensitized rabbit RBCs. The line marked with filled squares representsthe murine MAb 166-32. The line marked with filled circles representschimeric MAb 166-32. The line marked with filled triangles representsisotype-matched negative control antibody G3-519. The Y-axis represents% inhibition of hemolysis. The X-axis represents the antibodyconcentration.

FIG. 14 shows the inhibition of alternative pathway (AP) hemolysis ofunsensitized rabbit RBCs. The line marked with filled squares representschimeric 166-32 IgG. The line marked with filled circles representscFab/9aa. The line marked with filled triangles represents cFab. TheY-axis represents % inhibition of hemolysis. The X-axis represents theprotein concentration of the IgG and Fab.

FIG. 15 shows the effects of anti-factor D MAb 166-32 treatment on thehemodynamic functions of isolated rabbit hearts perfused with humanplasma. Left ventricular end-diastolic pressure (LVEDP) is representedby filled circles (for MAb 166-32) and filled squares (for MAb G3-519).Left ventricular developed pressure (LVDP) is represented by opencircles (for MAb 166-32) and open squares (for MAb G3-519). MAb G3-519is the isotype-matched irrelevant control.

FIG. 16 is a typical representation of left ventricular developedpressure (LVDP) by the two antibody groups in an isolated rabbit heartstudy. The upper panel represents a heart treated with the negativecontrol antibody, MAb G3-519, and the lower panel a heart treated withMAb 166-32. The MAb G3-519 treated heart was not able to maintain LVDPafter challenge with 4% human plasma, while the MAb 166-32 treated heartretained almost baseline LVDP after 60 minutes of perfusion with 4%human plasma.

FIG. 17 shows the concentration of Bb in lymphatic effluents at selectedtimepoints from isolated rabbit hearts perfused with 4% human plasma.Samples from MAb 166-32 treated hearts (open circles) containedsignificantly less Bb than MAb G3-519 treated hearts (filled squares),p<0.05.

FIG. 18 shows the alternative pathway hemolytic activity of plasmasamples collected at different time points from the extracorporealcircuits treated with MAb 166-32 (filled squares) or MAb G3-519 (filledcircles).

FIG. 19 shows the concentration of C3a in plasma samples collected atdifferent time points from the extracorporeal circuits treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 20 shows the concentration of sC5b-9 in plasma samples collected atdifferent time points from the extracorporeal circuits treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 21 shows the concentration of Bb in plasma samples collected atdifferent time points from the extracorporeal circuits treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 22 shows the concentration of C4d in plasma samples collected atdifferent time points from the extracorporeal circuits treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 23 shows the level of expression of CD11b on the surface ofneutrophils obtained at different time points from the extracorporealcircuits treated with MAb 166-32 (filled squares) or MAb G3-519 (filledcircles). The level of expression of CD11b is represented by meanfluorescence intensity (MFI) obtained by immunocytofluorometricanalyses.

FIG. 24 shows the level of expression of CD62P on the surface ofplatelets obtained at different time points from the extracorporealcircuits treated with MAb 166-32 (filled squares) or MAb G3-519 (filledcircles). The level of expression of CD62P is represented by meanfluorescence intensity (MFI) obtained by immunocytofluorometricanalyses.

FIG. 25 shows the concentration of neutrophil-specific myeloperoxidase(MPO) in plasma samples collected at different time points from theextracorporeal circuits treated with MAb 166-32 (filled squares) or MAbG3-519 (filled circles).

FIG. 26 shows the complete, selective inhibition of the alternativecomplement activity in plasma obtained from the extracorporeal circuitstreated with MAb 166-32 as compared to the negative control MAb G3-519.MAb 166-32 did not inhibit the classical complement activity of thehuman plasma in the circuits. The number of paired circuits is 5. Mean±S.E.M.; P=0.002 (paired T test) between MAb 166-32 and MAb G3-519circuits.

FIG. 27 shows that MAb 166-32 (closed squares) inhibits the productionof Bb in the extracorporeal circuits as compared to the negative controlMAb G3-519 (open squares). The number of paired circuits is 5. Mean±S.E.M.; P=0.001 (*) and P=0.0001 (**) by two-way ANOVA.

FIG. 28 shows that MAb 166-32 (closed squares) inhibits the productionof C4d in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The number of paired circuits is 5.Mean ±S.E.M. No significant difference between the two groups by two-wayANOVA.

FIG. 29 shows that MAb 166-32 (closed squares) inhibits the productionof C3a in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The number of paired circuits is 5.Mean ±S.E.M.; P=0.0003 (*) and P=0.0001 (**) by two-way ANOVA.

FIG. 30 shows that MAb 166-32 (closed squares) inhibits the productionof sC5b-9 in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The number of paired circuits is 5.Mean ±S.E.M.; P=0.01 (*) and P=0.0001 (**) by two-way ANOVA.

FIG. 31 shows that MAb 166-32 (closed squares) inhibits the productionof C5a in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The number of paired circuits is 5.Mean ±S.E.M.; P=0.003 (*), P=0.002 (**) and P=0.0001 (***) by two-wayANOVA.

FIG. 32 shows that MAb 166-32 (closed squares) inhibits the expressionof CD11b in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The level of CD11b expression isexpressed as fluorescent intensity normalized with reference to thevalue at time 0 (100%). The number of paired circuits is 5. Mean±S.E.M.; P=0.02 (*), P=0.01 (**), P=0.007 (***), P=0.003 (***) andP=0.0001 (****) by two-way ANOVA.

FIG. 33 shows that MAb 166-32 (closed squares) inhibits the productionof myeloperoxidase in the extracorporeal circuits as compared to thenegative control MAb G3-519 (open squares). The number of pairedcircuits is 5. Mean ±S.E.M.; P=0.004 (*) and P=0.0002 (**) by two-wayANOVA.

FIG. 34 shows that MAb 166-32 (closed squares) inhibits the productionof elastase-□1-antitrypsin in the extracorporeal circuits as compared tothe negative control MAb G3-519 (open squares). The number of pairedcircuits is 5. Mean ±S.E.M.; P=0.0007 (*) and P=0.0001 (**) by two-wayANOVA.

FIG. 35 shows that MAb 166-32 (closed squares) inhibits the expressionof CD62P on platelets in the extracorporeal circuits as compared to thenegative control MAb G3-519 (open squares). The level of CD62Pexpression is expressed as fluorescent intensity normalized withreference to the value at time 0 (100%). The number of paired circuitsis 5. Mean ±S.E.M.; P=0.0001 (*) by two-way ANOVA.

FIG. 36 shows that MAb 166-32 (closed squares) reduces the percentage ofplatelets expressing CD62P as compared to the negative control MAbG3-519 (open squares). The number of paired circuits is 5. Mean ±S.E.M.;P=0.05 (*), P=0.005 (**) and P=0.0001 (***) by two-way ANOVA.

FIG. 37 shows that MAb 166-32 (closed squares) inhibits the productionof platelet thrombospondin in the extracorporeal circuits as compared tothe negative control MAb G3-519 (open squares). The number of pairedcircuits is 5. Mean ±S.E.M.; P=0.016 (*), P=0.003 (**) and P=0.0001(***) by two-way ANOVA.

FIG. 38 shows that MAb 166-32 (closed squares) inhibits the productionof IL-8 in the extracorporeal circuits as compared to the negativecontrol MAb G3-519 (open squares). The number of paired circuits is 5.Mean ±S.E.M.; P=0.0001 (*) by two-way ANOVA.

FIG. 39 shows the pharmacokinetics of Mab 166-32 in a baboon model ofcardiopulmonary bypass (CPB).

FIG. 40 shows the inhibition of alternative complement activation by Mab166-32 in baboon CPB.

FIG. 41 shows the Bb concentration as affected by Mab 166-32.

FIG. 42 shows the C4d concentration as affected by Mab 166-32.

FIG. 43 shows the C3a concentration as affected by Mab 166-32.

FIG. 44 shows CD11b expression on neutrophils as affected by Mab 166-32.

FIG. 45 shows CD11b expression on monocytes as affected by Mab 166-32.

FIG. 46 shows CD62P expression on platelets as affected by Mab 166-32.

FIG. 47 shows plasma IL-6 levels as affected by Mab 166-32.

FIG. 48 shows plasma LDH levels as affected by Mab 166-32.

FIG. 49 shows plasma creatine kinase levels as affected by Mab 166-32.

FIG. 50 shows plasma creatine kinase levels as affected by Mab 166-32.

FIG. 51 shows plasma creatinine levels as affected by Mab 166-32.

FIG. 52 shows dynamic lung compliance of baboons in CPB as affected byMab 166-32.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 shows the nucleotide sequence of human factor D.

SEQ ID NO:2 shows the amino acid sequence of human factor D.

SEQ ID NO:3 shows the nucleotide sequence of pig factor D.

SEQ ID NO:4 shows the amino acid sequence of pig factor D.

SEQ ID NO:5 is the primer used for cloning the V_(K) gene of MAb 166-32.

SEQ ID NO:6 was used as an anealed adaptor for cloning the V_(K) gene ofMAb 166-32, as described below, and as a primer.

SEQ ID NO:7 was also used as an anealed adaptor for cloning the V_(K)gene of MAb 166-32, as described below.

SEQ ID NO:8 was used as a 3′ primer for cloning the V_(H) gene of MAb166-32, as described below.

SEQ ID NO:9 was a primer used for cloning the V_(H) gene of MAb 166-32.

SEQ ID NO:10 was used as a primer for cloning the V_(H) gene of MAb166-32.

SEQ ID NO:11 was the 5′ primer for the PCR of the Fd gene of MAb 166-32.

SEQ ID NO:12 was the 3′ primer for the PCR of the Fd gene of MAb 166-32.SEQ ID NO: 13 was the 5′ primer for the Fd gene PCR of MAb 166-32.

SEQ ID NO: 14 was the 3′ primer for the Fd gene PCR of MAb 166-32.

SEQ ID NO: 15 is the sequence for additional amino acids added to Fd toobtain recombinant Fab.

MAKING AND USING THE INVENTION

A. Generation of Monoclonal Antibodies (MAbs) to Human Factor D

In one embodiment of the invention, anti-factor D MAbs can be raised byimmunizing rodents (e.g. mice, rats, hamsters and guinea pigs) witheither native factor D purified from human plasma or urine, orrecombinant factor D or its fragments expressed by either eukaryotic orprokaryotic systems. Other animals can be used for immunization, e.g.non-human primates, transgenic mice expressing human immunoglobulins andsevere combined immunodeficient (SCID) mice transplanted with human Blymphocytes. Hybridomas can be generated by conventional procedures byfusing B lymphocytes from the immunized animals with myeloma cells (e.g.Sp2/0 and NS0), as described by G. Köhler and C. Milstein (Nature, 1975:256: 495-497). In addition, anti-factor D antibodies can be generated byscreening of recombinant single-chain Fv or Fab libraries from human Blymphocytes in phage-display systems. The specificity of the MAbs tohuman factor D can be tested by enzyme linked immunosorbent assay(ELISA), Western immunoblotting, or other immunochemical techniques. Theinhibitory activity of the antibodies on complement activation can beassessed by hemolytic assays using unsensitized rabbit or guinea pig redblood cells (RBCs) for the alternative pathway, and using sensitizedchicken or sheep RBCs for the classical pathway. The hybridomas in thepositive wells are cloned by limiting dilution. The antibodies arepurified for characterization for specificity to human factor D by theassays described above.

If used in treating inflammatory or autoimmune diseases in humans, theanti-factor D antibodies would preferably be used as chimeric,deimmunized, humanized or human antibodies. Such antibodies can reduceimmunogenicity and thus avoid human anti-mouse antibody (HAMA) response.It is preferable that the antibody be IgG4, IgG2, or other geneticallymutated IgG or IgM which does not augment antibody-dependent cellularcytotoxicity (S. M. Canfield and S. L. Morrison, J. Exp. Med., 1991:173: 1483-1491) and complement mediated cytolysis (Y. Xu et al., J.Biol. Chem., 1994: 269: 3468-3474; V. L. Pulito et al., J. Immunol.,1996; 156: 2840-2850).

Chimeric antibodies are produced by recombinant processes well known inthe art, and have an animal variable region and a human constant region.Humanized antibodies have a greater degree of human peptide sequencesthan do chimeric antibodies. In a humanized antibody, only thecomplementarity determining regions (CDRs) which are responsible forantigen binding and specificity are animal derived and have an aminoacid sequence corresponding to the animal antibody, and substantiallyall of the remaining portions of the molecule (except, in some cases,small portions of the framework regions within the variable region) arehuman derived and correspond in amino acid sequence to a human antibody.See L. Riechmann et al., Nature, 1988; 332: 323-327; G. Winter, U.S.Pat. No. 5,225,539; C. Queen et al., U.S. Pat. No. 5,530,101.

Deimmunized antibodies are antibodies in which the T and B cell epitopeshave been eliminated, as described in International Patent ApplicationPCT/GB98/01473. They have no immunogenicity, or reduced immunogenicity,when applied in vivo.

Human antibodies can be made by several different ways, including by useof human immunoglobulin expression libraries (Stratagene Corp., LaJolla, Calif.) to produce fragments of human antibodies (VH, VL, Fv, Fd,Fab, or F(ab′)₂, and using these fragments to construct whole humanantibodies using techniques similar to those for producing chimericantibodies. Human antibodies can also be produced in transgenic micewith a human immunoglobulin genome. Such mice are available fromAbgenix, Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J.

One can also create single peptide chain binding molecules in which theheavy and light chain Fv regions are connected. Single chain antibodies(“ScFv”) and the method of their construction are described in U.S. Pat.No. 4,946,778. Alternatively, Fab can be constructed and expressed bysimilar means (M. J. Evans et al., J. Immunol. Meth., 1995; 184:123-138). All of the wholly and partially human antibodies are lessimmunogenic than wholly murine MAbs, and the fragments and single chainantibodies are also less immunogenic. All these types of antibodies aretherefore less likely to evoke an immune or allergic response.Consequently, they are better suited for in vivo administration inhumans than wholly animal antibodies, especially when repeated orlong-term administration is necessary. In addition, the smaller size ofthe antibody fragment may help improve tissue bioavailability, which maybe critical for better dose accumulation in acute disease indications.

Based on the molecular structures of the variable regions of theanti-factor D antibodies, one could use molecular modeling and rationalmolecular design to generate and screen small molecules which mimic themolecular structures of the binding region of the antibodies and inhibitthe activities of factor D. These small molecules can be peptides,peptidomimetics, oligonucleotides, or organic compounds. The mimickingmolecules can be used as inhibitors of complement activation ininflammatory indications and autoimmune diseases. Alternatively, onecould use large-scale screening procedures commonly used in the field toisolate suitable small molecules form libraries of combinatorialcompounds.

In one preferred embodiment of the invention, a chimeric Fab, havinganimal (mouse) variable regions and human constant regions is usedtherapeutically. The Fab is preferred because:

it is smaller than a whole immunoglobulin and may provide better tissuepermeation;

as monovalent molecule, there is less chance of immunocomplexes andaggregates forming; and

it can be produced in a microbial system, which can more easily bescaled-up than a mammalian system.

B. Applications of the Anti-Factor D Molecules

The anti-factor D binding molecules, antibodies, and fragments of thisinvention can be administered to patients in an appropriatepharmaceutical formulation by a variety of routes, including, but notlimited, intravenous infusion, intravenous bolus injection, andintraperitoneal, intradermal, intramuscular, subcutaneous, intranasal,intratracheal, intraspinal, intracranial, and oral routes. Suchadministration enables them to bind to endogenous factor D and thusinhibit the generation of C3b, C3a and C5a anaphylatoxins, and C5b-9.

The estimated preferred dosage of such antibodies and molecules isbetween 10 and 500 μg/ml of serum. The actual dosage can be determinedin clinical trials following the conventional methodology fordetermining optimal dosages, i.e., administering various dosages anddetermining which is most effective.

The anti-factor D molecules can function to inhibit in vivo complementactivation and/or the alternative complement pathway and inflammatorymanifestations that accompany it, such as recruitment and activation ofmacrophages, neutrophils, platelets, and mast cells, edema, and tissuedamage. These inhibitors can be used for treatment of diseases orconditions that are mediated by excessive or uncontrolled activation ofthe complement system. These include, but are not limited to: (1) tissuedamage due to ischemia-reperfusion following acute myocardialinfarction, aneurysm, stroke, hemorrhagic shock, crush injury, multipleorgan failure, hypovolemic shock and intestinal ischemia; (2)inflammatory disorders, e.g., burns, endotoxemia and septic shock, adultrespiratory distress syndrome, cardiopulmonary bypass, hemodialysis;anaphylactic shock, severe asthma, angioedema, Crohn's disease, sicklecell anemia, poststreptococcal glomerulonephritis and pancreatitis; (3)transplant rejection, e.g., hyperacute xenograft rejection; and (4)adverse drug reactions, e.g., drug allergy, IL-2 induced vascularleakage syndrome and radiographic contrast media allergy. Autoimmunedisorders including, but not limited to, systemic lupus erythematosus,myasthenia gravis, rheumatoid arthritis, Alzheimer's disease andmultiple sclerosis, may also be treated with the inhibitors of theinvention.

The anti-factor D molecules can also be used diagnostically to ascertainthe presence of, or to quantify, factor D in a tissue specimen or a bodyfluid sample, such as serum, plasma, urine or spinal fluid. In thisapplication, well known assay formats can be used, such asimmunohistochemistry or ELISA, respectively. Such diagnostic tests couldbe useful in determining whether certain individuals are eitherdeficient in or overproduce factor D.

C. Animal Models of the Therapeutic Efficacy of Factor D Inhibitors

The therapeutic activity of factor D inhibitors in various diseaseindications described above can be confirmed by using available animalmodels for various inflammatory and autoimmune manifestations. The invitro tests described below in the examples are adequate to establishtheir efficacy.

Animal models relevant to various complement-related conditions anddiseases in humans can also be used to confirm the in vivo efficacy offactor D inhibitors. These conditions and diseases include myocardialischemia/reperfusion injury (H. F. Weisman et al., Science, 1990; 249:146-151); myocardial infarction (J. W. Homeister et al., J. Immunol.1993; 150: 1055-1064), systemic lupus erythematosus andglomerulonephritis (S. K. Datta. Meth. Enzymol., 1988; 162: 385-442; D.J. Salvant and A. V. Cybulsky, Meth. Enzymol., 1988; 162: 421-461),rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad. Sci., 1995; 92:8955-8959), adult respiratory distress syndrome (R. Rabinovici et al.,J. Immunol., 1992; 149: 1744-1750), hyperacute rejection in organtransplantation (T. J. Kroshus et al., Transplantation, 1995; 60:1194-1202), burn injury (M. S. Mulligan et al., J. Immunol., 1992; 148:1479-1485) and cardiopulmonary bypass (C. S. Rinder et al., J. Clin.Invest., 1995; 96: 1564-1572).

Exemplification of how to make and use the invention, and verificationof its utility, appear below.

EXAMPLE 1 Generation of Anti-Factor D MAbs

Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, were injectedsubcutaneously with 25 μg of factor D purified from human serum(Advanced Research Technologies, San Diego, Calif.) in complete Freund'sadjuvant (Difco Laboratories, Detroit, Mich.) in 200 μl ofphosphate-buffered saline (PBS) pH7.4. The factor D preparation weretested to be >95% pure by sodium dodecylsulphate (SDS)-polyacrylamidegel electrophoresis (PAGE). The factor D was tested and found to bebiologically active in hemolysis as described below. At two-weekintervals the mice were twice injected subcutaneously with 25 μg ofhuman factor D in incomplete Freund's adjuvant. Then two weeks later andthree days prior to sacrifice, the mice were again injectedintraperitoneally with 25 μg of the same antigen in PBS. For eachfusion, single cell suspensions were prepared from the spleen of animmunized mouse and used for fusion with Sp2/0 myeloma cells. 5×10⁸ ofthe Sp2/0 and 5×10⁸ spleen cells were fused in a medium containing 50%polyethylene glycol (M. W. 1450) (Kodak, Rochester, N.Y.) and 5%dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells werethen adjusted to a concentration of 1.5×10⁵ spleen cells per 200 μl ofthe suspension in Iscove medium (Gibco, Grand Island, N.Y.),supplemented with 10% fetal bovine serum, 100 units/ml of penicillin,100 μg/ml of streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin and16 μM thymidine. Two hundred microliters of the cell suspension wereadded to each well of about twenty 96-well microculture plates. Afterabout ten days culture supernatants were withdrawn for screening forreactivity with purified factor D in ELISA.

Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtestplates were coated by adding 50 μl of purified human factor at 50 ng/mlovernight at room temperature. The low concentration of factor D forcoating enabled the selection of high-affinity antibodies. After thecoating solution was removed by flicking of the plate, 200 μl of BLOTTO(non-fat dry milk) in PBS was added to each well for one hour to blockthe non-specific sites. An hour later, the wells were then washed with abuffer PBST (PBS containing 0.05% Tween 20). Fifty microliters ofculture supernatants from each fusion well were collected and mixed with50 μl of BLOTTO and then added to the individual wells of the microtestplates. After one hour of incubation, the wells were washed with PBST.The bound murine antibodies were then detected by reaction withhorseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fcspecific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) anddiluted at 1:2,000 in BLOTTO. Peroxidase substrate solution containing0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.) and 0.0003%hydrogen peroxide (Sigma) was added to the wells for color developmentfor 30 minutes. The reaction was terminated by addition of 50 μl of 2MH₂SO₄ per well. The OD at 450 nm of the reaction mixture was read with aBioTek ELISA Reader (BioTek Instruments, Winooski, VM).

The culture supernatants from the positive wells were then tested by twoassays: i) inhibition of alternative pathway hemolysis of unsensitizedrabbit RBCs by pre-titered human serum by the method described below;and ii) inhibition of formation of C3a by zymosan treated with humanserum, as described below. The cells in those positive wells were clonedby limiting dilution. The MAbs were tested again for reactivity withfactor D in the ELISA. The selected hybridomas were grown in spinnerflasks and the spent culture supernatant collected for antibodypurification by protein A affinity chromatography. Four MAbs were testedto be strongly reactive with human factor D in ELISA. These MAbs aredesignated 166-11, 166-32, 166-188, and 166-222 (FIG. 1). Among them,MAb 166-32 (IgG1) strongly inhibited the alternative pathway hemolysisof unsensitized rabbit RBCs as described below.

EXAMPLE 2 Determination the Kinetic Constants of the Anti-Factor D MAbsby Surface Plasmon Resonance Method

The kinetic constants for the binding of MAbs 166-11, 166-32, 166-188,and 166-22 to human factor D were determined by surface plasmonresonance-based measurements using the BIAcore instrument (PharmaciaBiosensor AB, Uppsala, Sweden). All the binding measurements wereperformed in HEPES-buffered saline (HBS) (10 mM HEPES, pH 7.4, 150 mMNaCl, 3.4 mM EDTA, 0.005% Surfactant P20) at 25° C. To measure thebinding rate constants of factor D to the MAbs, a rabbit anti-mouse IgG(H+L) was immobilized onto a CM5 sensorchip by amine coupling usingN-hydroxysuccinimide and N-ethyl-N′-(3-diethylaminopropyl) carbodimide.Each individual MAb was then captured onto the coated sensorchip beforethe injection of factor D at different concentrations. To measure theassociation rate constants (k_(assoc)), five dilutions of factor D (2.5nM, 5 nM, 10 nM, 15 nM and 20 nM) were made based on the concentrationindicated by the manufacturer, and were injected to the flowcell at theflow rate of 5 μl/min. To measure the dissociation rate constants(k_(dissoc)), 100 nM factor D was injected into the flowcell at the flowrate of 5 μl/min. The data, in the form of sensorgrams, was analyzedusing the data-fitting programs implemented in the BIAcore system. SinceMAb 166-32 has a very fast k_(assoc) which is beyond the reliabilitylimit of the assay format due to the limitation of the mass transporteffect, an additional binding format was also used to measure itskinetic rate constants. Factor D was immobilized onto the sensorchip byamine coupling as described above while MAb 166-32 of differentdilutions (5 nM, 10 nM, 15 nM, 20 nM and 25 nM for the measurement ofk_(assoc) and 200 nM for the measurement of k_(dissoc)) flowed to thesensorchip at the flow rate of 5 μl/min. The data, in the form ofsensorgrams, was analyzed as described above. The kinetic constants offactor D binding to the MAbs on BIAcore is shown in Table 1 below. MAbs166-32 and 166-222 have a very high affinity for factor D, withequilibrium dissociation constant (K_(D)) less than 0.1 nM.

TABLE 1 Kinetic Constants of Factor D Binding to MAbs on BIAcore MAbsk_(assoc) (×10⁵ M⁻¹ s⁻¹) k_(dissoc) (×10⁻⁴ s⁻¹) K_(D) (×10⁻¹⁰M)^(c)166-32^(a) >10 1.1 <1 166-32^(b) 4.6 0.76 1.6 166-188^(a) 8.75 2.1 2.4166-11^(a) 8.0 1.0 1.24 ^(a)Factor D was used as the analyte whichflowed onto the sensorchip coated with anti-factor D MAb captured byrabbit anti-mouse IgG. ^(b)MAb 166-32 was used as the analyte and factorD was crosslinked to the sensorchip by the amine-coupling method.^(c)K_(D), equilibrium dissociation constant, = k_(dissoc)/k_(assoc)

EXAMPLE 3 Inhibition of Complement-Activated Hemolysis

To study the functional activity of the anti-factor D MAbs in inhibitingcomplement activation in vitro, two hemolytic assays were used.

For the alternative pathway, unsensitized rabbit RBCs were washed threetimes with gelatin/veronal-buffered saline (GVB/Mg-EGTA) containing 2 mMMgCl₂ and 1.6 mM EGTA. EGTA at a concentration of 10 mM was used toinhibit the classical pathway (K. Whaley et al., in A. W. Dodds (Ed.),Complement: A Practical Approach. Oxford University Press, Oxford, 1997,pp. 19-47). The washed cells were re-suspended in the same buffer at1.7×10⁸ cells/ml. In each well of a round-bottom 96-well microtestplate, 50 μl of normal human serum (20%) was mixed with 50 μl ofGVB/Mg-EGTA or a serially diluted test MAb, then 30 μl of the washedrabbit RBCs suspension were added to the wells containing the mixtures.Fifty microliters of normal human serum (20%) was mixed with 80 μl ofGVB/Mg-EGTA to give the serum color background. For negative control, anisotype-matched anti-HIV-1 gp120 MAb, G3-519, was used. The finalmixture was incubated at 37° C. for 30 minutes. The plate was shaken ona micro-test plate shaker for 15 seconds. The plate was then centrifugedat 300×g for 3 minutes. 80 μl of the supernatants were collected andtransferred to wells on a flat-bottom 96-well microtest plates formeasurement of OD at 405 nm. The percent inhibition of hemolysis isdefined as 100×[(OD without MAb−OD serum color background)−(OD withMAb−OD serum color background)]/(OD without MAb−OD serum colorbackground).

FIG. 2 shows that MAb 166-32 strongly inhibits in a dose-dependentmanner the alternative pathway hemolysis of unsensitized rabbit RBCs inthe presence of 10% human serum, whereas the irrelevant isotype-matchedcontrol MAb G3-519 does not. MAb G3-519 is specific to the HIV envelopeglycoprotein gp120.

In assays to test the inhibitory activity of MAb166-32 in 90% humanserum, frozen human serum was thawed and pre-treated with EGTA at afinal concentration of 10 mM. Ten microliters of serially diluted MAb166-32 or G3-519 were added to 90 μl of EGTA-treated human serum induplicate wells of a 96-well microtest plate for 15 minutes at roomtemperature. Thirty microliters of the washed rabbit RBC's were added toeach well. The plate was incubated at 37° C. for 30 minutes. The platewas shaken on a plate-shaker for 15 seconds and then centrifuged at300×g for 3 minutes. Supernatants (80 μl) were collected and transferredto wells on a flat-bottom 96-well microtest plate for measurement of ODat 405 nm. Each plate contained two wells containing 100 μl of 90% humanserum and 30 μl of the buffer as serum color background and also twowells containing the RBCs lysed with 100 μl of 90% human serum, in theabsence of monoclonal antibody, to represent total lysis. FIG. 3 showsthat MAb 166-32 strongly inhibits in a dose-dependent manner thealternative pathway hemolysis of unsensitized rabbit RBCs even in thepresence of 90% human serum.

For the classical pathway, chicken RBCs (5×10⁷ cells/ml) ingelatin/veronal-buffered saline (GVB⁺⁺) containing 0.5 mM MgCl₂ and 0.15mM CaCl₂ were sensitized with purified rabbit anti-chicken RBCimmunoglobulins at 8 μg/ml (Inter-Cell Technologies, Hopewell, N.J.) for15 minutes at 4° C. The cells were then washed with GVB⁺⁺. The finalhuman serum concentration used was 2%.

FIG. 4 shows that MAb 166-32 and the irrelevant control G3-519 do notinhibit the classical pathway hemolysis of sensitized chicken RBCs,whereas the positive control anti-human C5 MAb 137-76 does. The datafrom FIGS. 2, 3 and 4 indicate that MAb 166-32 is specific to theinhibition of the alternative pathway of complement activation.

EXAMPLE 4 Specificity of MAb 166-32 to Factor D

Two hemolytic assays, as described below, were used to demonstrate thespecificity of MAb 166-32 to human factor D.

(1) Inhibition of Factor D Dependent Hemolytic Assays Using UnsensitizedRabbit RBCs

A human serum sample was first depleted of factor D by passing itthrough an affinity column packed with 3M Emphaze Biosupport Medium(Pierce, Rockford, Ill.) coupled with the anti-factor D MAb 166-222. Theflow-through serum was tested to be inactive in triggering alternativepathway hemolysis due to the complete depletion of factor D. Theprocedure of this assay is similar to that described in Example 3described above, except purified factor D of varying concentrations wasadded to the factor D depleted serum to reconstitute the hemolyticactivity. Under these conditions, the hemolysis of rabbit RBCs wasfactor D dependent. It was shown that the reconstituted hemolyticactivity is linearly proportional to the concentration of thesupplemented factor D (from 0.01 μg/ml to 2 μg/ml (FIG. 5)). The datafrom FIG. 5 also shows that 0.3 μg/ml of MAb 166-32 can completelyinhibit hemolysis of unsensitized rabbit RBCs in the presence of 0.1μg/ml supplemented factor D, whereas the negative control MAb G3-519 hasno effect on the factor D dependent hemolysis. These data suggest thatMAb 166-32 can effectively inhibit the biological activity of humanfactor D at a molar ratio of 1:2 (MAb 166-32 to factor D). Therefore MAb166-32 is a potent, high-affinity antibody to factor D. The antibody hasthe potential to be used clinically to treat diseases or conditionsrelating to activation of the alternative complement pathway.

(2) Inhibition of the Formation of Alternative C3 Convertase on EAC3BCells

EAC3b cells are sheep RBCs coated with human C3b (purchased from theNational Jewish Center of Immunology and Respiratory Medicine, Denver,Colo.). In this assay, the alternative C3 convertase was assembled onthe surface of EAC3b cells by addition of factor B, factor P (properdin)and factor D. EAC3b cells (5×10⁸), were then washed three times inDGVB⁺⁺ medium (50% veronal buffered saline, pH7.2, containing 0.075 mMCaCl₂, 0.25 mM MgCl₂, 0.1% gelatin, 2.5% (w/v) dextrose and 0.01% sodiumazide). The washed cells were then resuspended in 1.5 ml of DGVB⁺⁺,factor P (30 μg) and factor B (20 μg). The concentrations of factor Pand factor B were pre-determined to be in excess. Fifty microliters ofthe cell suspension was added to each well of a round-bottom 96-wellmicrotest plate. Then 50 μl of a mixture of factor D (1.2 ng/ml) andserially diluted MAb 166-32 or MAb G3-519 was added to the wellscontaining to the cells for incubation for 15 minutes at 30° C. Theconcentration of factor D (1.2 ng/ml) was pre-determined to give over90% hemolysis under these conditions. After incubation, the cells werewashed twice in GVB-EDTA medium (gelatin/veronal-buffered salinecontaining 10 mM EDTA). The cells were then resuspended in 30 μl ofGVB-EDTA medium. To initiate hemolysis, 100 μl of guinea pig serum(Sigma) (diluted 1:10 in GVB-EDTA) were added to each well. The mixtureswere then incubated at 37° C. for 30 minutes. The microtest plate wasthen centrifuged at 300×g for 3 minutes. The supernatant was collectedfor OD measurement at 405 nm.

FIG. 6 shows the results. It can be seen that MAb 166-32 inhibits thelysis of EAC3b cells, whereas the irrelevant MAb G3-519 does not. MAb166-32 inhibits factor D) from cleavage of factor B, thereforepreventing the formation of C3 convertase on the surface of EAC3b cells.

EXAMPLE 5 Inhibition of the Generation of C3a from Complement-ActivatedZymogen by MAb 166-32

To further ascertain the functional specificity of MAb 166-32 to factorD, the effect of the MAb on alternative complement activation on zymosan(activated yeast particles) was examined. Zymosan A (from Saccharomycescerevisiae, Sigma) (1 mg/ml) was washed three times in GVB/Mg-EGTA andthen resuspended in the same medium at 1 mg/ml. Twenty-five microlitersof MAb 166-32 or G3-519 in different concentrations were mixed with 25μl of human serum (diluted 1:5 in GVB/Mg-EGTA) in a microtube andincubated for 15 minutes at room temperature. The blank contained noantibody; only the plain medium and the serum. After incubation, 50 μlof washed zymosan suspension were added to each tube for incubation for30 minutes at 37° C. The microtubes were then centrifuged at 2000×g for5 minutes, the supernatants were collected and mixed with equal volumeof Specimen Stabilizing Solution (Quidel, San Diego, Calif.). Thesamples were frozen at −25° C. until being assayed. The concentration ofC3a and sC5b-9 in the samples were measured by quantitative ELISA kits(Quidel) according to the procedures provided by the manufacturer.

FIG. 7 shows that MAb 166-32 inhibits the generation of C3a fromcomplement-activated zymosan, whereas the irrelevant MAb G3-519 has noeffect. These data suggest that MAb 166-32 inhibits the formation of C3convertase by factor D. The complete inhibition of factor D by MAb166-32 can effectively block the formation of C3 convertase, asindicated by the inability to generate C3a. This will lead to theinhibition of C5 convertase in the subsequent steps of the complementcascade, as evidenced by the inhibition of sC5b-9 (MAC) formation (FIG.8).

EXAMPLE 6 Inhibition of Complement-Activated Hemolysis by the Fab of MAb166-32

In order to examine whether the monovalent form of MAb 166-32 is aseffective in inhibiting the alternative complement pathway as theparental bivalent MAb 166-32, the Fab of MAb 166-32 was prepared bypapain digestion using a commercial reagent kit (Pierce). The Fab wasthen tested for inhibitory activity on the alternative pathway hemolysisusing unsensitized rabbit RBCs as described above.

FIG. 9 shows that both the whole IgG and Fab show similar potency inblocking the alternative complement activation, taking intoconsideration that there are two binding sites per antibody molecule.These results suggest that monovalent form of MAb 166-32 is active andit retains similar potency against factor D as its parental bivalentantibody. A possible advantage of using the monovalent form as atherapeutic product is that they it may exhibit better tissuepenetration because of their smaller size. In view of the fact that theFab of MAb 166-32 is active, it is likely that the particular bindingepitope on factor D recognized by the MAb is functionally important.

EXAMPLE 7 Effects of MAb 166-32 on Alternative Pathway Hemolysis UsingSera from Different Animal Species

In order to study the cross-reactivity of MAb 166-32 with factor D fromdifferent animal species, alternative pathway hemolytic assays wereperformed using sera from different animal species. Fresh sera fromdifferent animal species (human, rhesus monkey, chimpanzee, baboon,cynomolgus monkey, sheep, dog, mouse, hamster, rat, rabbit, guinea pigand pig) were first tested for the CH50 values, which are defined as thedilution of the serum to achieve 50% lysis of unsensitized rabbit RBCs.The inhibitory activity of MAb 166-32 on the same hemolytic activity(CH50) of each serum was then tested and compared.

FIG. 10 shows that MAb 166-32 has strong inhibitory activity againstsera from human, rhesus monkey, cynomolgus monkey and chimpanzee andmoderate inhibitory activity against sera from baboon, cynomolgusmonkey, sheep and dog. The antibody does not inhibit sera from mouse,hamster, rat, rabbit, guinea pig and pig. These data suggests that MAb166-32 binds to an epitope on factor D shared by humans, rhesus monkeys,chimpanzees, baboons, cynomolgus monkeys, sheep and dogs.

EXAMPLE 8 Construction of Human Factor D Mutants for Epitope Mapping ofMAb 166-32

To delineate the binding epitope on human factor D recognized by MAb166-32, the reactivity of the antibody with human factor D on Westernblots was first tested. MAb 166-32 did not react with SDS-denaturedhuman factor D (either reduced or non-reduced) immobilized onnitrocellulose membrane. This result indicates the MAb 166-32 bindsnative but not denatured factor D.

Since MAb 166-32 does not inhibit the hemolytic activity of mouse andpig factor D as described in Example 7, it is likely that MAb 166-32binds to a site on human factor D that has a high degree of differencein the amino acid sequence from those of mouse and pig factor D. Basedon this concept, various factor D mutants and hybrids were made byreplacing amino acid residues in human factor D with the correspondingamino acid residues in the pig counterpart, for mapping the bindingepitope of Mab 166-32, as described below.

(1) Construction of Factor D Mutants and Hybrids

Human factor D gene segments were obtained by polymerase chain reaction(PCR) using human adipocyte cDNA (Clontech, San Francisco, Calif.) asthe template and appropriate oligonucleotide primers. Amplified DNAfragments were digested with BamHI and EcoRI restriction enzymes and thedigested product was inserted at the BamHI and EcoRI sites of theBaculovirus transfer vector pVL1393 (Pharmingen, San Diego, Calif.) togive the wild type pVL1393-factor D/Hu. The human factor D is designatedas factor D/Hu. The nucleotide sequence and the deduced amino acidsequence of the mature human factor D protein are shown in SEQ ID NOS: 1and 2 (R. T. White et al., J. Biol. Chem., 1992; 267:9210-9213; GenBankaccession number: M84526).

Pig factor D cDNA clone pMon24909 was obtained as a gift from J. L.Miner of University of Nebraska (GenBank accession number: U29948). TheBamHI-EcoRI fragments of pMon24909 were cloned into pVL1393 to givepVL1393-factor D/Pig. The pig factor D is designated as factor D/Pig.The nucleotide sequence and the deduced amino acid sequence of themature pig factor D protein are shown in SEQ ID NOS: 3 and 4.

Three human factor D mutants were constructed by using appropriateprimers and overlapping PCR. The amino acid mutations were designed byreplacing the amino acid residues in the human sequence with thecorresponding amino acid residues of the pig sequence, from thealignment of the amino acid sequences of human and pig factor D forhomology comparison. The first mutant, factor D/VDA, contained threeamino acid mutations, at position 113, Valine was substituted forGlutamic acid (designated, in shorthand, “V113E”), as well assubstitutions D116E and A118P. The second mutant, factor D/RH, containedtwo amino acid mutations: R156L and H159Y. The third mutant, factor D/L,contained a single mutation: L168M. DNA sequences encoding these mutantswere confirmed by DNA sequencing. After digesting with appropriateenzymes, DNA fragments were inserted at the BamHI and EcoRI sites of theBaculovirus transfer vector pVL1393 to give pVL1393-factorD/VDA,pVL1393-factorD/RH and pVL1393-factorD/L, respectively.

Two chimeric human-pig factor D hybrids were also constructed by usingappropriate primers and overlapping PCR. The first hybrid, factorD/Hupig, contained 52 human factor D-derived amino acids at theN-terminus, and the remaining amino acids were derived from the pigfactor D. The other hybrid, factor D/Pighu, contained 52 pig factorD-derived amino acids at the N-terminus and the remaining amino acidswere derived from the human factor D. The BamHI and EcoRI-digested DNAfragments were inserted at the BamHI and EcoRI sites of the Baculovirustransfer vector pVL1393 to give pVL1393-factorD/Hupig andpVL1393-factorD/Pighu.

(2) Expression of Factor D Mutants and Hybrids

The procedures for transfection of the plasmids, generation ofrecombinant Baculoviruses, and production of the recombinant factor Dproteins in insect cells Sf9 were done according to the manufacturer'smanual (Baculovirus Expression Vector System, Pharmingen).

(3) Purification of Factor D Mutants and Hybrids

Factor D mutant and hybrid proteins from the culture supernatants ofinfected Sf9 cells were purified by affinity chromatography usingpurified sheep anti-human factor D polyclonal antibodies (The BindingSite Limited, San Diego, Calif.). Three milliliters of sheep anti-humanfactor D antibodies (13.2 mg/ml) were equilibrated in a coupling buffer(0.1 M borate and 0.75 M Na₂SO₄, pH 9.0) and coupled with 4 ml ofUltralink Biosupport Medium (Pierce) for 2 hours at room temperature.The beads were washed first with 50 mM diethylamine, pH 11.5 to saturateall the remaining reactive sites and then with a buffer containing 10 mMTris, 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, and 0.02% NaN₃, pH 8.0.The gel was stored in the buffer at 4° C.

Culture supernatant harvested from 100 ml spinner culture of Sf9 cellsinfected with the various Baculovirus mutants were passed through thesheep anti-factor D affinity column which was pre-equilibrated with PBSto remove the storage buffer. The bound factor D proteins were elutedwith 50 mM diethylamine, pH 11.5. The collected fractions wereimmediately neutralized to pH 7.0 with 1 M Hepes buffer. Residual saltswere removed by buffer exchange with PBS by Millipore membraneultrafiltration (M.W. cut-off: 3,000) (Millipore Corp., Bedford, Mass.).Protein concentrations were determined by the BCA method (Pierce).

(4) Factor D ELISA

The reactivity of MAb 166-32 with the various factor D mutants andhybrids was tested by ELISA. Different wells of 96-well microtest plateswere coated with the proteins (factor D/Hu, factor D/Pig, factorD/Hupig, factor D/Pighu, factor D/VDH, factor D/RH, and factor D/L) byaddition of 100 μl of each protein at 0.5 μg/ml in PBS. After overnightincubation at room temperature, the wells were treated with PBSTB (PBSTcontaining 2% BSA) to saturate the remaining binding sites. The wellswere then washed with PBST. One hundred microliters of serially dilutedMab 166-32 (1 μg/ml to 0.5 ng/ml) were added to the wells for 1 hour atroom temperature. The wells were then washed with PBST. The boundantibody was detected by incubation with diluted HRP-goat anti-mouse IgG(Fc) (Jackson ImmunoResearch) for 1 hour at room temperature. Peroxidasesubstrate solution was then added for color development as describedabove. The OD was measured using an ELISA reader at 450 nm.

FIG. 11 shows that MAb 166-32 reacts with factor D/Hu, factor D/Pighu,and factor D/VDA, but not factor D/Pig, factor D/Hupig, factor D/RH, andfactor D/L. The ELISA results indicate that amino acid residues Arg156,His159 and Leu168 of human factor D are essential for the binding of MAb166-32. This is consistent with the fact that MAb 166-32 did not bindfactor D/Hupig when the C-terminus portion of human factor D wasreplaced with that of pig. Amino acid residues Arg156, His159 and Leu168are located in a so-called “methionine loop” constituted by a disulfidelinkage between Cys154 and Cys170 with a methionine residue at position

169 (J. E. Volanakis et al., In: The Human Complement System in Healthand Disease, J. E. Volanakis and M. M. Franks, eds., Marcel Dekker,1998, pp. 49-81). Structurally, the “methionine loop” is a member of arigid type 1 β turn. It is found to be exposed on the surface of thefactor D molecule based on the data from X-ray crystallography studies(S. V. L. Narayana et al., J. Mol. Biol., 1994, 235: 695-708). However,the contribution of the “methionine loop” to substrate specificity andcatalysis of factor D has never been studied (J. E. Volanakis et al.,Protein Sci., 1996; 5: 553-564). The data here have demonstrated for thefirst time that the “methionine loop” plays an important role in thefunctional activity of factor D. MAb 166-32 and its Fab, when bound tothis region on factor D, can effectively inhibit the catalysis of factorB.

EXAMPLE 9 Cloning of Anti-Factor D MAb 166-32 Variable Region Genes andConstruction and Expression of Chimeric 166-32 IgG and its Fab

In order to reduce the immunogenicity of MAb 166-32 when used in humans,a chimeric form of MAb 166-32 was made by replacing the mouse constantregions with human constant regions of IgG1. Two forms of chimeric Fabof the antibody were also made by replacing the mouse constant regionswith their human counterparts. The cloning of MAb 166-32 variable regiongenes and the construction and expression of the chimeric 166-32antibody and its Fab are described below.

(1) Cloning of Anti-Factor D MAb Variable Region Genes

Total RNA was isolated from the hybridoma cells secreting anti-Factor DMAb 166-32 using RNAzol following the manufacturer's protocol (Biotech,Houston, Tex.). First strand cDNA was synthesized from the total RNAusing oligo dT as the primer. PCR was performed using the immunoglobulinconstant (C) region-derived 3′ primers and degenerate primer setsderived from the leader peptide or the first framework region of murineV_(H) or V_(K) genes as the 5′ primers. Although amplified DNA was notedfor V_(H), no DNA fragment of expected lengths was amplified for V_(K).Both V_(H) and V_(K) genes were cloned by anchored PCR.

Anchored PCR was carried out as described by Chen and Platsucas (Scand.J. Immunol., 1992; 35: 539-549). For cloning the V_(K) gene,double-stranded cDNA was prepared using the NotI-MAK1 primer(5′-TGCGGCCGCTGTAGGTGCTGTCTTT-3′ SEQ ID NO:5). Annealed adaptors AD1(5′-GGAATTCACTCGTTATTCTCGGA-3′ SEQ ID NO:6) and AD2(5′-TCCGAGAATAACGAGTG-3′ SEQ ID NO:7) were ligated to both 5′ and 3′termini of the double-stranded cDNA. Adaptors at the 3′ ends wereremoved by NotI digestion. The digested product was used as the templatein PCR with the AD1 oligonucleotide as the 5′ primer and MAK2(5′-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3′ SEQ ID NO:8) as the 3′ primer. DNAfragments of approximately 500 bp were cloned into pUC19. Twelve cloneswere selected for further analysis. Seven clones were found to containthe CDR3 sequence specific for the Sp2/0 V message, and presumably werederived from the aberrant κ light chain messages of the fusion partnerfor the 166-32 hybridoma cell line. The NotI-MAK1 and MAK2oligonucleotides were derived from the murine Cκ region, and were 182and 84 bp, respectively, downstream from the first bp of the Cκ gene.Three clones were analyzed by DNA sequencing, yielding sequencesencompassing part of murine Cκ, the complete Vκ, and the leader peptide.

For cloning the V_(H) gene, double-stranded cDNA was prepared using theNotI-MAG1 primer (5′-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3′ SEQ ID NO:9).Annealed adaptors AD1 and AD2 were ligated to both 5′ and 3′ termini ofthe double-stranded cDNA. Adaptors at the 3′ ends were removed by NotIdigestion. The digested product was used as the template in PCR with theAD1 oligonucleotide and MAG2 (5′-CGGTAAGCTTCACTGGCTCAGGGAAATA-3′ SEQ IDNO:10) as primers. DNA fragments of 500 to 600 bp in length were clonedinto pUC19. The NotI-MAG1 and MAG2 oligonucleotides were derived fromthe murine Cγ 1 region, and were 180 and 93 bp, respectively, downstreamfrom the first bp of the murine Cγ 1 gene. Three clones were analyzed byDNA sequencing, yielding sequences encompassing part of murine Cγ 1, thecomplete V_(H), and the leader peptide.

(2) Construction of Expression Vectors for Chimeric 166-32 IgG and Fab

The V_(H) and Vκ genes were used as templates in PCR for adding theKozak sequence to the 5′ end and the splice donor to the 3′ end. Afterthe sequences were analyzed to confirm the absence of PCR errors, theV_(H) and Vκ genes were inserted into expression vector cassettescontaining human Cγ 1 and Cκ, respectively, to give pSV2neo V_(H)-huCγ1and pSV2neoV-huCκ. CsC1 gradient-purified plasmid DNAs of the heavy- andlight-chain vectors were used to transfect COS cells by electroporation.After 48 hours, the culture supernatant was tested by ELISA to containapproximately 200 ng/ml of chimeric IgG. The cells were harvested andtotal RNA was prepared. First strand cDNA was synthesized from the totalRNA using oligo dT as the primer. This cDNA was used as the template inPCR to generate the Fd and κ DNA fragments. For the Fd gene, PCR wascarried out using

5′-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3′ (SEQ ID NO:11) as the 5′primer and a CH1-derived 3′ primer (5′-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3′ SEQ ID NO: 12). The DNA sequence was confirmed tocontain the complete V_(H) and the CH1 domain of human IgG1. Afterdigestion with the proper enzymes, the Fd DNA fragments were inserted atthe HindIII and BamHI restriction sites of the expression vectorcassette pSV2dhfr-TUS to give pSV2dhfrFd (FIG. 12A).

For the κ gene, PCR was carried out using5′-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3′ (SEQ ID NO:13) as the 5′primer and a Cκ-derived 3′ primer (5′-CGGGATCCTTCTCCCTCTAACACTCT-3′ SEQID NO:14). DNA sequence was confirmed to contain the complete Vκ andhuman Cκ regions. After digestion with proper restriction enzymes, the κDNA fragments were inserted at the Hind III and BamHI restriction sitesof the expression vector cassette pSV2neo-TUS to give pSV2neoκ (FIG.12B). The expression of both Fd and κ genes are driven by theHCMV-derived enhancer and promoter elements. Since the Fd gene does notinclude the cysteine amino acid residue involved in the inter-chaindisulfide bond, this recombinant chimeric Fab contains non-covalentlylinked heavy- and light-chains. This chimeric Fab is designated as cFab.

To obtain recombinant Fab with an inter-heavy and light chain disulfidebond, the above Fd gene was extended to include the coding sequence foradditional 9 amino acids (EPKSCDKTH SEQ ID NO: 15) from the hinge regionof human IgG1. The BstEII-BamHI DNA segment encoding 30 amino acids atthe 3′ end of the Fd gene was replaced with DNA segments encoding theextended Fd. The sequence of the extended Fd with an additional 9 aminoacids from the hinge region of human IgG1 was confirmed by DNAsequencing. This Fd/9aa gene was inserted into the expression vectorcassette pSV2dhfr-TUS to give pSV2dhfrFd/9aa. This chimeric Fab isdesignated as cFab/9aa.

(3) Expression of Chimeric 166-32 IgG and Fab

To generate cell lines secreting chimeric 166-32 IgG, NS0 cells weretransfected with purified plasmid DNAs of pSV2neoV_(H)-huCγ1 andpSV2neoV-huCκ by electroporation. Transfected cells were selected in thepresence of 0.7 mg/ml G418. Cells were grown in a 250-ml spinner flaskusing serum-containing medium.

To generate cell lines secreting chimeric 166-32 Fab, CHO cells weretransfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa)and pSV2neoκ by electroporation. Transfected cells were selected in thepresence of G418 and methotrexate. Selected cell lines were amplified inincreasing concentrations of methotrexate. Cells were single-cellsubcloned by limiting dilution. High-producing single-cell subclonedcell lines were then grown in 100-ml spinner culture using serum-freemedium.

(4) Purification of Chimeric 166-32 IgG

Culture supernatant of 100 ml spinner culture was loaded on a 10-mlPROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column waswashed with 10 bed volumes of PBS. The bound antibody was eluted with 50mM citrate buffer, pH 3.0. Equal volume of 1 M Hepes, pH 8.0 was addedto the fraction containing the purified antibody to adjust the pH to7.0. Residual salts were removed by buffer exchange with PBS byMillipore membrane ultrafiltration (M.W. cut-off: 3,000). The proteinconcentration of the purified antibody was determined by the BCA method(Pierce).

(5) Purification of Chimeric 166-32 Fab

Chimeric 166-32 Fab was purified by affinity chromatography using amouse anti-idiotypic MAb to MAb 166-32. The anti-idiotypic MAb isdesignated as MAb 172-25-3. It was made by immunizing mice with MAb166-32 conjugated with keyhole limpet hemocyanin (KLH) and screening forspecific MAb 166-32 binding could be competed with human factor D.

The affinity chromatography matrix was prepared by mixing 25 mg MAb172-25-3 with 5 ml of dry azlactone beads (UltraLink Biosupport Medium,Pierce) in a coupling buffer (0.1 M borate and 0.75 M Na₂SO₄, pH 9.0)for 2 hours at room temperature. Then the residual reactive sites wereblocked with 1 M ethanolamine, pH 9.0 for 2.5 hours at room temperature.The beads were then washed in a buffer containing 10 mM Tris, 0.15 MNaCl, 5 mM EDTA, 1% Triton X-100 and 0.02% NaN₃, (pH 8.0) and storedthen at 4° C.

For purification, 100 ml of supernatant from spinner cultures of CHOcells producing cFab or cFab/9aa were loaded onto the affinity columncoupled with MAb 172-25-3. The column was then washed thoroughly withPBS before the bound Fab was eluted 50 mM diethylamine, pH 11.5.Residual salts were removed by buffer exchange as described above. Theprotein concentration of the purified Fab was determined by the BCAmethod (Pierce).

(6) SDS-PAGE of Chimeric 166-32 IgG, cFab and cFab/9aa

The purified chimeric 166-32 IgG, cFab and cFab/9aa were analyzed forpurity and molecular size by SDS-PAGE. The proteins were treated withsample buffers with or without mercaptoethanol. The samples were thenrun on pre-cast gels (12.5%) (Amersham Pharmacia Biotech, Uppsala,Sweden) together with pre-stained molecular weight standard (lowmolecular weight range) (BIO-RAD Laboratories, Hercules, Calif.) usingthe PhastSystem (Amersham Pharmacia Biotech). The gels were then stainedin Coomassie Brilliant Blue solution (BIO-RAD) for 5 minutes and thende-stained in an aqueous solution containing 40% methanol and 10% aceticacid.

The results of an SDS-PAGE of cFab, cFab/9aa and chimeric IgG treatedunder both non-reducing and reducing condition, showed chimeric IgG hasa protein band of 150 kD, and two protein bands of heavy (HC) and light(LC) chains of approximately 50 kD and 29 kD, respectively. As expected,cFab/9aa had only 1 protein band of about 40 kD under non-reducingcondition, indicating that the heavy and light chains are linked by aninter-chain disulfide bond. On the other hand, cFab showed two proteinbands under non-reducing condition, indicating that the heavy and lightchains are not linked by an inter-chain disulfide bond.

(7) Determination of the Activities of Chimeric 166-32 IgG, cFab andcFab/9aa

The activities of chimeric 166-32 IgG, cFab and cFab/9aa were determinedby using the alternative complement hemolytic assay described above.FIG. 13 shows that the murine and chimeric forms of MAb 166-32 haveidentical potency in inhibiting factor D. FIG. 14 shows that cFab andcFab/9aa have almost identical potency in inhibiting factor D. Mostimportantly, the potency of the two forms of chimeric Fab is identicalto that of the chimeric IgG, taking into consideration that there aretwo binding sites per IgG molecule. Together, the results demonstratethat chimeric IgG, cFab and cFab/9aa retain the potency of the parentalmurine MAb 166-32.

EXAMPLE 10 Protection of Complement-Mediated Tissue Damage by Mab 166-32in an Ex Vivo Model of Rabbit Hearts Perfused with Human Plasma

Activation of the complement system contributes to hyperacute rejectionof xenografts. It may occur as a result of binding of complement fixingantibodies, the direct activation of complement via the alternativepathway on foreign cell surfaces, and/or the failure of complementregulation by the foreign organ (J. L. Platt et al., Transplantation,1991; 52: 937-947). Depending on the particular species-speciesinteraction, complement activation through either the classical oralternative pathway predominates, though in some cases both pathways maybe operative (T. Takahashi et al., Immunol. Res., 1997; 16: 273-297).Previous studies have shown that hyperacute rejection can occur in theabsence of anti-donor antibodies via activation of the alternativepathway (P. S. Johnston et al., Transplant. Proc., 1991; 23: 877-879).

To demonstrate the importance of the alternative complement pathway intissue damage, the anti-factor D MAb 166-32 was tested using an ex vivomodel in which isolated rabbit hearts were perfused with diluted humanplasma. This model was previously shown to cause damage to the rabbitmyocardium due to the activation of the alternative complement pathway(M. R. Gralinski et al., Immunopharmacology, 1996: 34: 79-88).

(1) Langendorff Perfused Rabbit Hearts:

Male, New Zealand White rabbits (2.2-2.4 kg) were euthanized by cervicaldislocation. The hearts were removed rapidly and attached to a cannulafor perfusion through the aorta. The perfusion medium consisted of arecirculating volume (250 ml) modified Krebs-Henseleit (K-H) buffer (pH7.4, 37° C.) delivered at a constant rate of 20-25 ml/min. Thecomposition of the buffer medium in millimoles per liter was as follows:NaCl, 117; KCl, 4.0; CaCl₂. H₂O, 2.4; MgCl₂.6H₂O, 1.2; NaHCO₃, 25;KH₂PO₄, 1.1; glucose, 5.0; monosodium L-glutamate, 5.0; sodium pyruvate,2.0; and BSA, 0.25% (w/v). The K-H buffer passed through a gas porous“lung” consisting of Silastic™ Laboratory Grade Tubing (Dow Corning,Midland, Mich.), 55.49 meters in length, with an inner diameter of 1.47mm and an outer diameter of 1.96 mm. The membranous “lung” was exposedcontinuously to a mixture of 95% O₂/5% CO₂ to obtain an oxygen partialpressure within the perfusion medium equal to 500 mm Hg. The hearts werepaced throughout the protocol via electrodes attached to the rightatrium. Pacing stimuli (3 Hz, 4 msec duration) were delivered from alaboratory square wave generator (Grass SD-5, Quincy, Mass.). Thepulmonary artery was cannulated with polyethylene tubing to facilitatecollection of the pulmonary artery effluent, representing the coronaryvenous return. The superior and inferior vena cava and the pulmonaryveins were ligated to prevent exit of the perfusate from the severedvessels. A left ventricular drain, thermistor probe, and latex balloonwere inserted via the left atrium and positioned in the left ventricle.The fluid-filled latex balloon was connected with rigid tubing to apressure transducer to permit for measurement of left ventricularsystolic and end-diastolic pressures. The left ventricular developedpressure is defined as the difference between the left ventricularsystolic and end-diastolic pressures. The intraventricular balloon wasexpanded with distilled water to achieve an initial baseline leftventricular end-diastolic pressure of 5 mm Hg. Coronary perfusionpressure was measured with a pressure transducer connected to a side-armof the aortic cannula. All hemodynamic variables were monitoredcontinuously using a multichannel recorder (Grass Polygraph 79D, Quincy,Mass.). The isolated hearts were maintained at 37° C. throughout theexperimental period by enclosing them in a temperature-regulateddouble-lumen glass chamber and passing the perfusion medium through aheated reservoir and delivery system.

(2) Antibody Treatments:

Two treatment groups were used to determine the ability of anti-factor DMAb 166-32 to inhibit the effects of complement activation in isolatedrabbit hearts perfused with human plasma. Group 1: Isotype-matchednegative group, consisted of hearts perfused with 4% human plasma in thepresence of 0.3 μg/ml of MAb G3-519 (n=6) specific to HIV-1 gp120. Group2: Treatment group, consisted of hearts perfused with 4% human plasma inthe presence of 0.3 μg/ml of MAb 166-32 (n=6). The human plasma wasseparated from freshly collected whole blood and stored at −80° C. untiluse. This percentage of human plasma was chosen because it severelyimpairs myocardial function over a reasonable length of time and allowsone to assess the efficacy of a treatment regimen. Higher concentrationsof human plasma in this system cause the heart to rapidly developcontracture, making it difficult to analyze the effects of a drug at alow concentration. Preliminary studies had determined that 0.3 μg/ml wasthe minimal effective concentration that could protect the isolatedheart from the effects of complement activation. All hearts underwent10-15 minutes of equilibration in the Langendorff apparatus beforeaddition of either antibody to the perfusion medium. Ten minutes afterthe addition of antibody, 4% human plasma was added to the perfusionmedium (250 ml, recirculating). Hemodynamic variables, includingcoronary perfusion pressure (CPP), left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure (LVEDP), and leftventricular developed pressure (LVDP) were recorded before the additionof antibody (baseline), before the addition of 4% human plasma, andevery 10 minutes thereafter, for 60 minutes.

MAb 166-32 (0.3 μg/ml) attenuated the increase in coronary perfusionpressure (CPP) when compared to hearts treated with MAb G3-519 (0.3μg/ml) when exposed to 4% human plasma. A rise in CPP indicates coronaryvascular resistance which is often associated with myocardial tissuedamage. Isolated rabbit hearts perfused with MAb 166-32 maintained leftventricular end-diastolic pressure (LVEDP), in marked contrast to theresults obtained with MAb G3-519 (FIG. 15). The latter group of heartsdeveloped a progressive increase in LVEDP after exposure to 4% humanplasma, indicating contracture or a failure of the ventricle to relaxduring diastole (FIG. 15). MAb 166-32 also attenuated the decrease inleft ventricular developed pressure (LVDP) compared to the heartstreated with MAb G3-519 after exposure to diluted human plasma. (FIG.15).

FIG. 16 depicts representative recordings of cardiac function obtainedbefore and after 10, 30 and 60 minutes of perfusion in the presence of4% human plasma. The progressive increase in the LVEDP and the decreasein the LVDP of a MAb G3-519 treated rabbit heart is obvious after 10minutes with progressive deterioration of ventricular function over thesubsequent 50 minutes. On the other hand, preservation of ventricularfunction of a heart treated with MAb 166-32 is evident over the periodof 60 minutes.

Taken together, the hemodynamic data indicate that anti-factor D MAb166-32 protects isolated rabbit hearts from human complement-mediatedinjury as manifested by an overall maintenance of myocardial functionafter challenge with human plasma.

(3) Complement Bb ELISA:

Factor D catalyzes the cleavage of bound factor B, yielding the Ba andBb fragments. The concentration of Bb present serves as an index offactor D activity. The concentrations of activated component Bb in thelymphatic fluid collected from the isolated rabbit hearts were measuredusing a commercially available ELISA kit (Quidel). The assays made useof a MAb directed against human complement Bb to measure the activationof human complement system during perfusion of rabbit hearts in thepresence of human plasma. Lymphatic effluent from the severed lymphaticvessels was collected from the apex of the heart, snap-frozen in liquidnitrogen, and stored at −70° C. until assayed. The flow rate of thelymphatic effluent was recorded and accounted for in order to normalizethe Bb concentration.

Ten minutes after perfusion with 4% human plasma and at every time pointthereafter, a significantly (p<0.05) lower concentration of Bb waspresent in lymphatic effluents from hearts treated with MAb 166-32 ascompared to hearts treated with MAb G3-519 (FIG. 17). The decrease inthe production of the complement activation product Bb in the MAb 166-32treated rabbit hearts confirms the inhibitory activity of the antibodyon factor D.

(4) Immunohistochemical Localization of C5b-9 Deposition:

At the completion of the protocol, hearts were removed from theLangendorff apparatus, cut into transverse sections, and frozen inliquid nitrogen. The apex and atrial tissues were discarded. Sectionswere embedded in O.C.T. compound embedding medium (Miles, Inc., Ekhart,Ind.), cut to 3 μm, and placed on poly-L-lysine coated slides. Afterrinsing with phosphate-buffered saline (PBS), sections were incubatedwith 4% paraformaldehyde in PBS at room temperature. Heart sections wererinsed with PBS and incubated with 1% BSA for 15 minutes to minimizenon-specific staining. After rinsing with PBS, sections were incubatedwith a murine anti-human C5b-9 MAb (Quidel) at a 1:1,000 dilution atroom temperature for 1 hour. Sections were rinsed with PBS again andthen incubated at room temperature for 1 hour with a goat anti-murineFITC conjugated antibody (Sigma) at a 1:320 dilution. After a finalrinse with PBS, sections were mounted with Fluoromount-G (ElectronMicroscopy Sciences, Fort Washington, Pa.) and protected with acoverslip. Controls included sections in which the primary antibody wasomitted and sections in which an isotype-matched murine antibody IgG1(Sigma) was substituted for the anti-C5b-9 MAb.

Heart sections from MAb 166-32 and MAb G3-519 treated hearts wereexamined for human MAC (or C5b-9) deposition by immunofluorescencestaining. MAb 166-32 treated hearts exhibited a reduction in MACdeposition as compared to MAb G3-519 treated hearts.

In all, the data from the ex vivo studies of rabbit hearts demonstratethe efficacy of MAb 166-32 in preventing cardiac tissue injury as aresult of the inhibition of the alternative complement pathway.Inhibition of complement activation has been shown to prolong thesurvival of xenografts (S. C. Makrides, Pharmacological Rev., 1998, 50:59-87). Therefore, MAb 166-32 could potentially be used as a therapeuticagent to protect xenografts from destruction by human plasma.

EXAMPLE 11 Inhibitory Effects of MAb 166-32 on Complement Activation andInflammatory Reactions in an Extracorporeal Circulation Model ofCardiopulmonary Bypass A Pilot Study in which Human Whole Blood fromDifferent Donors was Used for Each Individual Circuit

Patients undergoing cardiopulmonary bypass (CPB) frequently manifest ageneralized systemic inflammatory response syndrome. Clinically, thesereactions are reflected in postoperative leukocytosis, fever, andextravascular fluid accumulation which may lead to prolonged recoveryand occasionally with serious organ dysfunction (J. K. Kirklin et al.,J. Thorac. Cardiovasc. Surg., 1983; 86: 845-857; L. Nilsson et al.,Scand. J. Thorac. Cardiovasc. Surg., 1988; 22: 51-53; P. W.

Weerwind et al., J. Thorac. Cardiovasc. Surg., 1995; 110: 1633-1641).The inflammatory responses consist of humoral and cellular changes thatcontribute to both tissue injury and impaired hemostasis. Complementactivation has been implicated as the important cause of the systemicinflammatory reaction (P. Haslam et al., Anaesthesia, 1980; 25: 22-26;A. Salama et al., N. Eng. J. Med., 1980; 318: 408-414; J. Steinberg etal., J. Thorac. Cardiovasc. Surg., 1993; 106: 1008-1016). Complementactivation is attributed to the interaction between the blood and thesurface of the extracorporeal circuit constituting CPB machines (D.Royston, J. Cardiothorac. Vasc. Anesth., 1997; 11: 341-354). Primaryinflammatory substances are generated after activation of the complementsystem, including the anaphylatoxins C3a and C5a, the opsonin C3b, andthe membrane attack complex C5b-9. C5a has been shown to upregulateCD11b (integrin) and CD18 (integrin) of MAC-1 complex inpolymorphonuclear cells PMN (comprising mainly neutrophils) in vitro (M.P. Fletcher et al., Am. J. Physiol., 1993; 265: H1750-H1761) and toinduce lysosomal enzyme release by PMN. C5b-9 can induce the expressionof P-selectin (CD62P) on platelets (T. Wiedmer et al., Blood, 1991; 78:2880-2886), and both C5a and C5b-9 induce surface expression ofP-selectin on endothelial cells (K. E. Foreman et al., J. Clin. Invest.,1994; 94: 1147-1155). C3a and C5a stimulate chemotaxis of human mastcells (K. Hartmann et al., Blood, 1997; 89: 2868-2870) and trigger therelease of histamine (Y. Kubota, J. Dermatol., 1992; 19: 19-26) whichinduces vascular permeability (T. J. Williams, Agents Actions, 1983; 13:451-455).

In vitro recirculation of whole blood in an extracorporeal bypasscircuit has been used extensively as a model to simulate leukocytes (J.Kappelamyer et al., Circ. Res., 1993; 72: 1075-1081; N. Moat et al.,Ann. Thorac. Surg., 1993; 56: 1509-1514; C. S. Rinder et al., J. Clin.Invest., 1995: 96: 1564-1572) and platelets (V. L. Jr. Hennesy et al.,Am. J. Physiol., 1977; 2132: H622-H628; Y. Wachtfogel et al., J. Lab.Clin. Med., 1985; 105: 601-607; C. S. Rinder et al., ibid) changes andcomplement activation (P. G. Loubser, Perfusion, 1987; 2: 219-222; C. S.Rinder et al., ibid; S. T. Baksaas et al., Perfusion, 1998; 13: 429-436)in CPB. The effectiveness of the anti-factor D MAb 166-32 to inhibit thecellular and complement activation in human whole blood was studiedusing this extracorporeal circulation model for CPB.

(1) Extracorporeal Circuit Preparation:

Extracorporeal circuits were assembled using a hollow-fiber pediatricmembrane oxygenator with an integrated heat exchanger module (D 901LILLPUT 1; DIDECO, Mirandola (MO), Italy), a pediatric venous reservoirwith an integrated cardiotomy filter (D 752 Venomidicard; DIDECO), aperfusion tubing set (Sorin Biomedical, Inc., Irvine, Calif.) and amultiflow roller pump (Stockert Instruments GmbH, Munich, Germany).Oxygenator and circuitry were primed with Plasma-Lyte 148 solution(Baxter Healthcare Corp., Deerfield, Ill.). The prime was warmed to 32°C. with a cooler-heater (Sarns; 3M Health Care, Ann Arbor, Mich.) andcirculated at 500 ml/min, while the sweep gas flow was maintained at0.25 liters per min using 100% oxygen. The sweep gas was changed to amixture of oxygen (95%) and carbon dioxide (5%) after the blood wasadded to the circuit. The pH, PCO₂, PO₂, and perfusate temperature werecontinuously monitored throughout the recirculation period. Sodiumbicarbonate was added as required to maintain pH in the range of7.25-7.40.

(2) Extracorporeal Circuit Operation and Sampling:

450 ml of blood were drawn over 5-10 minutes from healthy volunteers onno medications into a transfer pack (Haemo-Pak; Chartermed, Inc.,Lakewood, N.J.) containing porcine heparin (5 units/ml, finalconcentration; Elkins-Sinn, Cherry Hill, N.J.) and the anti-factor D MAb166-32 or the isotype-matched negative control MAb G3-519 (18 μg/mlfinal concentration). This concentration of antibody is equivalent toabout 1.5 times the molar concentration of factor D in the blood. Priorto the addition of the blood to the extracorporeal circuit, a bloodsample was taken from the transfer pack as the “pre-circuit” sample,designated the “−10 minute sample”. The blood was then added to thereservoir via the prime port. Prime fluid was simultaneously withdrawndistal to the oxygenator outlet to yield a final circuit volume of 600ml and a final hematocrit of 25-28%. Blood was circulated with prime,and complete mixing was accomplished within 3 minutes; a baseline samplewas drawn and designated as time 0. To mimic usual procedures ofsurgical operation under hypothermia, the circuit was then cooled to 27°C. for 70 minutes after which it was rewarmed to 37° C. for another 50minutes (for a total of 120 minutes of recirculation).

Blood samples were also drawn at 10, 25, 40, 55, 70, 80 and 120 minutesduring the recirculation. Plasma samples were prepared by immediatecentrifugation at 2,000×g at 4° C. Aliquots for the alternative pathwayhemolytic assays and neutrophil-specific myeloperoxidase assays weresnap-frozen on dry ice and then stored at −80° C. Aliquots formeasurement of complements C3a, C4d, sC5b-9, and Bb by ELISA wereimmediately mixed with equal volume of a Specimen Stabilizing Medium(Quidel), snap-frozen on dry ice, and then stored at −80° C. Samples ofwhole blood were also collected for immunostaining of the activationcell surface markers CD11b and CD62P on neutrophils and platelets,respectively. To prevent subsequent complement activation of the wholeblood samples during the staining procedure, 10 μl of 1 M EDTA wereadded to every ml of whole blood to give a final concentration of 10 mM.

(3) Alternative Pathway Hemolytic Assays:

The alternative complement activity in the plasma samples at differenttime points from the MAb 166-32 treated and the MAb G3-519 treatedcircuits were tested using rabbit red blood cells as described above.Fifty microliters of each sample (20%) were mixed with 50 μl ofGVB/Mg-EGTA buffer before addition of 30 μl of rabbit red blood cells(1.7×10⁸ cells/ml). After incubation at 37° C. for 30 minutes, thesupernatants were collected and OD read at 405 nm using an ELISA platereader.

FIG. 18 shows that the alternative complement activity in the MAb 166-32treated circuit was completely inhibited by the antibody, whereas MAbG3-519 had no effect on the complement activity when used in thecorresponding circuit. The results indicate MAb 166-32 is a potentinhibitor of the alternative complement pathway. Even at a molar ratioof only 1.5:1 (MAb:factor D), MAb 166-32 can completely inhibit thealternative complement activity.

(4) Assays of Complement Activation Products:

In addition to the hemolytic assays described above, the plasma samplesfrom the two extracorporeal circuits were tested for the levels of C3a,sC5b-9, Bb, and C4d. These substances were quantitated usingcommercially available ELISA kits (Quidel) according to themanufacturerer's manuals. Like C5a, sC5b-9 is an alternative marker forC5 convertase activity in the complement cascade. Both C5a and sC5b-9are produced as a result of cleavage of C5 by C5 convertase. ComplementBb is a specific marker for the activation of the alternative complementpathway, whereas C4d a specific marker for the activation of theclassical pathway.

FIGS. 19 and 20 show that MAb 166-32 inhibited effectively theproduction of C3a and sC5b-9 respectively, whereas the isotype-matchednegative control MAb G3-519 did not. The specificity and potency of MAb166-32 are further elucidated in FIGS. 21 and 22. The production of Bbby the alternative complement pathway was completely inhibited by MAb166-32, whereas the levels of Bb in the G3-519 circuit increase overtime during the recirculation. Interestingly, the levels of C4d in bothMAb 166-32 and G3-519 circuit did not vary significantly over time. Thelatter results on Bb and C4d levels strongly indicate that thecomplement activation in extracorporeal circulation is mediated mainlyvia the alternative pathway.

In sum, the results indicate that MAb 166-32 is a potent inhibitor ofthe alternative complement pathway. Inhibition of factor D can abolishthe complement activation in the subsequent steps of the cascade asmanifested by the reduction in C3a and sC5b-9 formation.

(5) Assays for the Activation of Neutrophils and Platelets:

The activation of neutrophils and platelets were quantitated bymeasuring the levels of the cell-surface expression of CD11b and CD62Pon neutrophils and platelets, respectively. For CD11b labeling ofneutrophils, 100 μl of whole blood collected from the circuits wereimmediately incubated with 20 μl of phycoerythrin (PE)-anti-CD11bantibody (clone D12, Becton Dickinson, San Jose, Calif.) for 10 minutesat room temperature in a microcentrifuge tube. Then 1.4 ml of FACSLysing Solution (Becton Dickinson) was added for 10 minutes at roomtemperature to lyse red blood cells and to fix leukocytes. Themicrocentrifuge tubes were centrifuged at 300×g for 5 minutes. Thesupernatant was aspirated and the cells resuspended in PBS for washing.The microcentrifuge tubes were spun again, the supernatant aspirated,and the cells finally resuspended in 0.5 ml of 1% paraformaldehydeovernight prior to analysis using an EPIC-XL flow cytometer (CoulterCorp., Miami, Fla.). For double labeling to concomitantly identify theneutrophil population, 5 μl of fluorescein isothiocyanate (FITC)-anti-CD15 antibody (clone MMA, Becton Dickinson) were added for incubationtogether with PE-anti-CD11b antibody.

For CD62P labeling of platelets, 40 μl of whole blood collected from thecircuits were immediately incubated with 20 μl of PE-anti-CD62P antibody(clone AC1-2, Becton Dickinson) for 10 minutes at room temperature in amicrocentrifuge tube. Then the mixture was treated with FACS LysingSolution as described above. The microcentrifuge tubes were centrifugedat 2,000×g for 5 minutes. The platelets were washed in PBS, fixed in 1%paraformaldehyde and then analyzed as described above. For doublelabeling to concomitantly identify the platelet population, 5 μl ofFITC-anti-CD42a antibody were added for incubation together withPE-anti-CD62P antibody.

For flow cytometric measurement, the PMN (containing mainly neutrophils)and platelet populations were identified by live-gating based onforward- versus side-scatter parameters and specific staining withFITC-anti-CD 15 antibody and FITC-anti-CD42a antibody, respectively. Thebackground staining was gated using isotype-matched labeled antibodies.The intensity of expression of CD11b and CD62P was represented by meanfluorescence intensity (MFI).

FIG. 23 shows that neutrophils from the MAb 166-32 treatedextracorporeal circuit showed substantially lower expression of CD11b ascompared to those from the MAb G3-519 treated circuit. These datatogether with the others above indicate that inhibition of thealternative complement activation by MAb 166-32 can prevent activationof neutrophils.

Similarly, FIG. 24 shows that platelets from the MAb 166-32 treatedextracorporeal circuit showed substantially lower expression of CD62P ascompared to those from the MAb G3-519 treated circuit. Again these datatogether with those above indicate that inhibition of the alternativecomplement activation by MAb 166-32 can prevent activation of platelets.

(6) Assay of Neutrophil-Specific Myeloperoxidase (MPO)

The degree of activation of neutrophils was also measured using acommercial ELISA kit (R & D Systems, Inc., Minneapolis, Minn.) toquantitate the amount of neutrophil-specific myeloperoxidase (MPO) inthe plasma samples from the extracorporeal circuits. MPO is stored inprimary granules (azurophilic) of neutrophils. It is released whenneutrophils undergo de-granulation during activation. Therefore MPO is asoluble marker for neutrophil activation. The assays were performedaccording to the manufacturer's manual. Briefly, samples were incubatedin the cells of a microplate, which have been coated with a first MAb toMPO. The MPO-MAb complex is labeled with a biotin-linked polyclonalantibody prepared from goat MPO-antisera. The final step of the assay isbased on a biotin-avidin coupling in which avidin has been covalentlylinked to alkaline phosphatase. The amount of MPO in each sample isenzymatically measured upon addition of the substrate4-nitrophenyl-phosphate (pNPP), by reading OD at 405 nm.

FIG. 25 shows that the levels of MPO in the MAb 166-32 treated circuitwere substantially lower than those in the MAb G3-519 circuit. Theresults corroborate those on the expression of CD11b in theimmunofluorometric studies described above.

Taken together, the data on complement, neutrophils and plateletssupport the notion that effective inhibition of the alternativecomplement activation in extracorporeal circulation by the anti-factor DMAb 166-32 can abolish the formation of inflammatory substances C3a, C5aand sC5b-9 and thus reduce the activation of neutrophils and platelets.Based on these results, it is anticipated that MAb 166-32, as well asits fragments, homologues, analogues and small molecule or peptidecounterparts thereof, will be effective in preventing or reducingclinical inflammatory reactions caused by CPB.

EXAMPLE 12 Inhibitory Effects of Mab 166-32 on Complement Activation andInflammatory Reactions in an Extracorporeal Circulation Model ofCardiopulmonary Bypass A Modified Study in which Whole Blood from theSame Donor was Used in Both Mab 166-32 and in Mab G3-519 (NegativeControl) Circuits on the Same Day

In order to avoid any variations in complement activity in the bloodfrom different donors, we modified the study design described in Example11. In this modified protocol, whole blood from the same donor was usedin both MAb 166-32 and MAb G3-519 circuits on the same day.

In the study, whole blood was obtained from 5 healthy subjects for fivepaired (test and control) circuits. Anti-factor D MAb 166-32 (4.5 mg)was added to 250 ml of freshly collected, heparinized human whole bloodto give a final concentration of 18 □g/ml (equivalent to about 1.5 timesthe molar concentration of human factor D in the blood). MAb G3-519 wasadded at the same final concentration to another 250 ml of heparinizedwhole blood from the same donor on the same day for a parallel circuit.The blood was then added to an extracorporeal circuit consisting of ahollow-fiber pediatric membrane oxygenator. The circuit was primed withlactated Ringer's solution which was circulated at 500 ml/minute. Theinitial volume of the diluted blood in the circuit was 350 ml. Thehematocrit was maintained at 26-28%. To simulate hypothermic CPBcommonly performed in open-heart surgery, the re-circulated blood wasmaintained at 27° C. for the initial 70 minutes; the circuits werere-warmed to 37° C. at 80 minutes. The temperature was maintained until120 min.

Blood samples were collected at different time points during there-circulation. Complement activation products (Bb, C4d, C3a, andsC5b-9), CD11b expression on neutrophils, CD62P expression on platelets,and neutrophil-specific myeloperoxidase were measured by the methodsdescribed in Example 11.

C5a was also measured quantitatively by an ELISA. Briefly, eightmicroliters of 1 M EDTA was added to eighty microliters of human plasmain an Eppendorf microfuge tube to prevent complement activation. Then 88□l of a precipitating reagent (Amersham, Arlington Heights, Ill.) wereadded to the sample for incubation at room temperature for 30 minutes.This procedure would precipitate high molecular weight proteins,including C5. The mixture was then centrifuged at 15,000×g for 15minutes at 4° C. For the C5a ELISA, protein A purified rabbit anti-C5apolyclonal antibodies (Calbiochem, La Jolla, Calif.) was used to coatwells of 96-well microtest plates to capture C5a in plasma, whereaspurified sheep anti-C5 polyclonal antibodies (Biodesign, Kennebunk, Me.)was used for detection. The bound sheep antibodies were detected byHRP-conjugated rabbit anti-sheep IgG (Fc) antibodies (JacksonImmunoResearch). Purified C5a (Advanced Research Technologies) was usedas the standard for calibration. In the assays, each well of 96-wellmicrotest plates was incubated with 100 μl of rabbit anti-C5a antibodies(2 μg/ml) overnight at room temperature. After the solution was removed,the remaining binding sites in the wells of the plastic plates weresaturated by incubation with 200 μl of PBSTB for 1 hour as describedabove. The wells were then washed with PBST. Treated plasma at serialdilutions was added to the wells in duplicate for 1 hour at roomtemperature. The wells were then washed again. One hundred μl of sheepanti-human C5 antibodies at 5 μg/ml were added to the well for 1 hour atroom temperature. The wells were then washed again. Then 100 μl ofdiluted HRP-rabbit anti-sheep IgG(Fc) antibodies were added for 1 hourat room temperature. After the wells were washed, peroxidase substratesolution was added for color development and OD₄₅₀ was measured asdescribed above.

A quantitative ELISA developed at Tanox was used to measure neutrophilspecific elastase-α1-antitrypsin complex. Upon activation, neutrophilrelease elastase is immediately complexed with α1-antitrypsin inhibitorin the blood. The assay used sheep anti-human neutrophil elastasepolyclonal antibodies (Biodesign) to immobilize the complex andHRP-conjugated sheep anti-1-antitrypsin inhibitor polyclonal antibodies(Biodesign) for detection. For a calibration standard, theelastase/α1-antitrypsin inhibitor complexes were made by mixingneutrophil-specific elastase (Elastin Products Company, Inc.,Owensville, Mo.) with α1-antitrypsin inhibitor (Calbiochem) at a ratioof 1:1 (w/w). In the ELISA, 100 μl of sheep anti-human neutrophilelastase (5 μg/ml) were added to each well of 96-well microtest platesfor incubation overnight at room temperature. After the solution wasremoved, the remaining binding sites on the plastic wells were saturatedby incubation with 200 μl of PBSTB for one hour at room temperature. Thewells were then washed. One hundred μl of serially diluted plasmasamples were added to each well in duplicate for incubation for 1 hourat room temperature. After the wells were washed, 100 μl ofHRP-conjugated sheep anti-α1-antitrypsin inhibitor antibodies (dilutedat 1:2,000) were added to each well for 1 hour at room temperature. Thewells were then washed and peroxidase substrate solution was added tothe well for color development. OD₄₅₀ was then measured as describedpreviously.

A quantitative ELISA was used to measure platelet thrombospondin whichis a soluble marker for platelet degranulation upon activation. In theassay, thrombospondin was captured by monoclonal anti-thrombospondin(clone P12) (Coulter) and then detected by biotinylated monoclonalanti-thrombospondin (clone 10) (Coulter) as described (A. E. Fiane etal., Immunopharmacol., 1999; 42: 231-243). Briefly, 100 μl of monoclonalanti-thrombospondin (clone P12) at 2 μg/ml was added to each well of96-well microtest plates and incubated overnight at room temperature.After the solution was removed, non-specific binding sites on theplastic wells were saturated by incubation with 200 μl of PBSTB for 1hour at room temperature. After the wells were washed, 100 μl ofserially diluted plasma was added to each well in duplicate. The wellswere then washed. One hundred μl biotinylated monoclonalanti-thrombospondin (clone P10) at 1 μg/ml was added to each well andincubated for 1 hour at room temperature. The wells were then washed.One hundred μl of streptavidin-conjugated HRP (Jackson ImmunoResearch)was added to each well and incubated for 1 hour at room temperature.After the wells were washed, peroxidase substrate solution was added forcolor development and OD450 was measured as described above. Purifiedthrombospondin (Calbiochem) was used as for a calibration standard inthe assay.

IL-8 was measured by an ELISA kit (R & D Systems). IL-8, aproinflammatory cytokine, is produced by neutrophils,monocytes/macrophages, and T cells upon activation by anaphylatoxins.

The data from 5 paired circuits were statistically analyzed using 2-wayANOVA of factorial, randomized block design. The data were representedas mean ±standard error.

(2) Results and Conclusions

MAb 166-32 at 18 μg/ml inhibited completely the alternative complementactivity measured by a hemolytic assay using unsensitized rabbit RBCs,whereas the negative control Mab G3-519 had no effect (FIG. 26). On theother hand, MAb 166-32 did not inhibit the classical pathway hemolysisof sensitized sheep RBCs. The selective inhibition of the alternativecomplement pathway is in agreement with the complete inhibition of theproduction of Bb (FIG. 27). Bb is the enzymatic product of factor Bactivation specific to the alternative complement pathway. On the otherhand, there was an increase in the plasma concentration of C4d in bothcircuits treated with either MAb 166-32 or G3-519 (FIG. 28). C4d is aspecific marker for activation of the classical complement pathway.

The plasma concentrations of C3a, sC5b-9 and C5a in the extracorporealcircuits were also measured. MAb 166-32 strongly inhibited theproduction of these substances (FIGS. 29, 30 and 31). Since thealternative complement activity of the plasma in the test circuits wascompletely inhibited by MAb 166-32, the production of C3a, sC5b-9 andC5a in these circuits could be attributed to some activation of theclassical pathway as indicated by the elevation of plasma C4d (FIG. 28).Activation of the classical pathway in extracorporeal circuits could betriggered by contact activation of the intrinsic coagulation pathway (T.E. Mollnes, Vox. Sang., 1998; 74 Suppl. 2: 303-307). As a result, factorXlla is released and binds C1-INH, an endogenous inhibitor of C1. Whenthe plasma concentration of C1-INH is reduced, C1 is more susceptible toactivation and thus augments the classical complement pathway. Inasmuchas the production of C5a and sC5b-9 were substantially inhibited ascompared to C3a, the moderate activation of the classical pathway asdescribed above did not seem to effectively activate C5 in this system.This is in agreement with the findings that the alternative complementpathway is predominantly activated in CPB (J. K. Kirklin et al., J.Thorac. Cardiovasc. Surg., 1983; 86: 845-857). Similarly althoughprotamine neutralization of heparin after CPB causes activation of theclassical complement pathway, the activation of C5 by protamine/heparincomplexes is not very effective (J. K. Kirklin et al., Ann. Thorac.Surg., 1986: 41: 193-199).

Activation of neutrophils as manifested by upregulation of surfaceexpression of CD11b was measured by immunocytofluorometric analyses. Theexpression of CD11b on neutrophils in circuits treated with MAb 166-32was significantly reduced as compared to the negative control MAb G3-519(FIG. 32). Reduction of CD11b expression on neutrophils will prevent theadhesion of neutrophils to platelets and endothelial cells via theinteraction with CD62P (P-selectin), an important hallmark ofneutrophil-mediated inflammation (C. Rinder et al., Blood, 1992; 79:1201-1205).

The activation of neutrophils was also studied by measuring the levelsof neutrophil-specific myeloperoxidase and elastase in plasma.Myeloperoxidase is stored in primary granules (azurophilic) ofneutrophils and released upon degranulation of activated neutrophils.Myeloperoxidase is responsible for production of hypochlorous acid(HOCl), a powerful oxidant that could cause tissue damage.Neutrophil-specific elastase is also released upon degranulation ofactivated neutrophils. The elastase could cause tissue injury as aresult of hydrolysis of elastin in extracellular matrices. Consistentwith the inhibition of neutrophil activation (FIG. 32), the release ofboth myeloperoxidase and elastase was also significantly inhibited byMAb 166-32 in the extracorporeal circuits (FIGS. 33 and 34).

The activation of platelets was inhibited by MAb 166-32 treatment of theextracorporeal circuits. The number of platelets expressing CD62P andthe level of CD62P expression level on platelets were significantlyreduced, respectively (FIGS. 35 and 36). The inhibition of plateletactivation in MAb 166-32 treated circuits is also manifested by thereduction in thrombospondin release from activated platelets (FIG. 37).

To further study the relationship between complement activation and theinflammatory responses in whole blood, we examined the plasma levels ofthe pro-inflammatory cytokine IL-8 in the circuits. The treatment ofhuman whole blood with MAb 166-32 reduced IL-8 production (FIG. 38).IL-8 is produced predominantly by activated neutrophils,monocytes/macrophages and T cells. IL-8 is closely associated withinflammatory diseases and CPB (M. C. Diago et al., Acta Anaesthesiol.Scand., 1997; 41: 725-730; S. S. Ashraf et al., J. Cardiothorac. Vascu.Anesthes., 1997; 11: 718-722). IL-8 causes chemotaxis of neutrophils, Tcells and basophils, degranulation of neutrophils, and adhesion ofneutrophils to endothelial cells.

Taken together, the results of the extracorporeal circulation studiesboth in Examples 11 and 12 show that complement activation playsimportant roles in inducing a plethora of cellular and humoralinflammatory responses. Anti-factor D MAb 166-32 is effective ininhibiting these inflammatory responses. Therefore complete inhibitionof the alternative pathway complement by Mab 166-32 is expected to bebeneficial to patients undergoing CPB.

EXAMPLE 13 Study of the Effects of Mab 166-32 in a Dog Model of Ischemiaand Reperfusion Injury

A study was designed to examine whether MAb 166-32 would protectmyocardial tissues from injury due to ischemia and reperfusion in dogs,although it was recognized at the outset that dog might not be adesirable animal model to study this indication for MAb 166-32. Theability of MAb 166-32 to neutralize dog factor D in hemolytic assays wasat least 10 times less effective as compared to human factor D (seeExample 7). Because of the limited amount of MAb 166-32 available at thetime, MAb 166-32 was administered into the heart via the intracoronaryblood vessel. It was hoped that the antibody would build up aconcentration of at least 60 μg/ml in the coronary blood in order toinhibit completely dog factor D in the heart. The dosage was calculatedto be 3.15 mg/kg/infusion for 6 infusions. MAb G3-519 was used as theisotype-matched control in the study.

Briefly, purpose-bred hound dogs were anaesthetized. A left thoracotomywas performed at the fourth intercostal space to expose the heart. Theproximal left circumflex coronary artery was isolated and ligated for 90minutes for induction of ischemia that was followed by 6 hours ofreperfusion. The antibody was given 6 times at 30 minutes beforeischemia, 10 minutes before reperfusion, and then 75, 150, 225, 300minutes during reperfusion. Radioactive microspheres were injected atdifferent time points to measure regional blood flow. At the end of theexperiments, hearts were perfused with Evans blue dye andtriphenyltetrazolium for measurement of area at risk and infarct size,respectively. Coronary lymph and whole blood from the jugular vein werecollected before ischemia and at the end of the experiment. Thesesamples were used to measure the concentration of the injectedantibodies and alternative pathway hemolytic activity.

The results show that the highest achievable concentration of MAb 166-32in the coronary lymph was about 30 μg/ml, which is well below theconcentration required for complete inhibition of dog factor D in thecoronary circulation. The antibody was also detected in the systemiccirculation, suggesting that the injected antibody dissipated outside ofthe heart. The data from the hemolytic assays show that alternativecomplement activity was not reduced; as is consistent with the fact thatthe concentration of the antibody was low. Therefore, it is not possibleto draw a conclusion on the effect of MAb 166-32 in reperfusion fromthese experiments in dogs.

EXAMPLE 14 Effects of Mab 166-32 in Baboons Undergoing CardiopulmonaryBypass

Anti-factor D Mab 166-32 (murine antibody) was studied in a baboon modelof hypothermic cardiopulmonary bypass (CPB) for pharmacokinetics and forits inhibitory effects on complement, neutrophils, monocytes, platelets,and tissue injury. Baboons was selected as the animal model because theyhave been extensively used as a non-human primate model in CPB studies(Hiramatsu Y et al., J. Lab. Clin. Med. 1997; 130: 412-420; Gikakis N etal., J. Thorac. Cardiovasc. Surg. 1998; 116: 1043-1051) and Mab 166-32is equally effective at inhibiting baboon factor D and human factor D.

Fourteen healthy adult baboons (ca. 15 kg of body weight) were used inthe study. The baboons were pre-screened for negative serologicalreactivity with Mab 166-32 by ELISA. The baboons were assigned to twogroups: 7 in the Mab 166-32-treated group and 7 in a saline-controlgroup. Baboons in the treatment group received a single intravenousbolus injection of Mab 166-32 at 5 mg/kg, whereas baboons in the controlgroup received an equal volume of saline. This dosage of Mab 166-32 waschosen because it was estimated to be adequate to completely neutralizethe factor D for about 7 hours, based on previously obtained data thatMab 166-32 at 1 mg/kg completely inhibited factor D in rhesus monkeysfor at least 1.5 hours. Mab 166-32 inhibited baboon, monkey and humanfactor D equally well.

Baboons were sedated with an intramuscular injection of 10 mg/kg ofketamine hydrochloride, and anesthesia was induced with 5 mg/kg ofthiopental sodium. The animals were then intubated, and generalanesthesia was maintained with inhaled isoflurane.

Heparin (300 units/kg) was administered for anti-coagulation. After amedian stemotomy was performed, the ascending aorta and the right atriumwere cannulated with a 14-French aortic cannular (DLP, Inc. GrandRapids, Mich., USA) and a 26-French single-stage venous cannula(Polystan A/S, Varlose, Denmark), respectively. Lactated Ringer'ssolution was then used to prime the extracorporeal circuit. The primingvolume for the whole circuit was approximately 600 ml. This particularlow-prime circuit was used to avoid the need for donor blood. During theCPB, the hematocrit was maintained at 26% to 28%. Pulmonary artery flowwas assessed with a 12-mm perivascular flow probe (Transonic Systems,Ithaca, N.Y., USA), before and after CPB, for cardiac outputmeasurements.

The CPB circuit consisted of a conventional non-pulsatile roller pump(Stockert, Irvine, Calif., USA), a hollow-fiber membrane oxygenator(Capiorx SX10; Terumo Corp., Tokyo, Japan), an arterial filter (TerumoCorp.), and silicone elastomer tubing (Dow Corning, Inc., Midland,Mich., USA). The pump flow rate was maintained at 80 ml/kg/min. DuringCPB, the mean arterial pressure was maintained at 50-60 mm Hg by addingisoflurane through the oxygenator inflow conduit. At the end of CPB,protamine (1 mg/100 units of heparin) was administered for heparinneutralization. The heart rate, systemic arterial pressure, centralvenous pressure, and pulmonary arterial pressure was continuouslymonitored. At the end of the experiment, each animal was euthanized withintravenous boluses of Beuthanasia-D (0.22 mg/kg).

Blood samples were taken from the animals for assays at different timepoints: Before injection of Mab 166-32 or saline (at 0 hour), afterinjection of Mab 166-32 or saline (at 45 minutes), before CPB (at 1hour), 10 minutes in CPB at 37° C., 25 minutes in CPB at 27° C., 85minutes in CPB at 27° C., 105 minutes in CPB at 37° C. after re-warming,135 minutes in CPB at 37° C. prior to protamine administration (at 3.25hours), 30 minutes after CPB at 37° C., 1 hour after CPB at 37° C., 2hours after CPB at 37° C., 6 hours after CPB at 37° C. (at 9.25 hours),and finally 18 hours after CPB at 37° C. (at 21.25 hours). Experimentswith 8 of the 14 baboons (4 in the treatment group and 4 in the controlgroup) were terminated at 6 hours after CPB, whereas the remaininganimals (3 in the treatment group and 3 in the control group) wereterminated 18 hours after CPB.

Plasma and whole blood samples were used in different assays to measure:

(1) Plasma concentration of free Mab 166-32 by ELISA using factor D asthe coating antigen;

(2) Functional activity of factor D in baboon plasma as determined bytwo hemolytic assays: rabbit red blood cells for the alternativecomplement pathway and sensitized chicken red blood cells for theclassical complement pathway;

(3) Plasma concentration of complement Bb, C4d, and C3a by ELISA (QuidelCorp., San Diego, Calif., USA);

(4) Expression level of CD11b on neutrophils and monocytes byimmunofluorocytometric methods;

(5) Expression level of CD62P on platelets by immunofluorocytometricmethods;

(6) Plasma concentration of IL-6 by ELISA (BioSource International,Inc., Camarillo, Calif., USA); and

(7) Plasma concentration of lactate dehydrogenase (LDH), creatine kinase(CK), creatine kinase MB isoenzymes (CK-MB), and creatinine.

The data were analyzed statistically by student's T-test (for C4d) and2-way ANOVA of repeated measurements (for other parameters) (significantat p<0.05). The data were represented as mean ±SEM.

Results and Discussion

In this study, baboons were treated with a single intravenous bolusinjection of 5 mg/kg of Mab 166-32. The plasma concentrations of thefree antibody were measured by an ELISA using human factor D as coatingantigen (FIG. 39). At 45 minutes after the antibody injection, theplasma concentration of free Mab 166-32 was 68.3±9.9 μg/ml. The antibodyconcentration then decreased to 23.4±4.4 μg/ml at 10 minutes after CPB,as a result of hemodilution upon the initiation of CPB. The antibodyconcentration remained at 10-13 μg/ml until 3 hours after CPB. Theantibody concentration reduced to 6.2±2.3 μg/ml at 6 hours after CPB andto 1.7±3.9 μg/ml at 18 hours after CPB.

Using an alternative complement hemolytic assay with rabbit red bloodcells, the functional activity of factor D in the plasma samples fromthe Mab166-32 treated animals was measured (FIG. 40). The alternativecomplement hemolytic activity of the baboon plasma samples wascompletely inhibited at 6 hours after CPB. At 18 hours after CPB, theinhibition was reduced to 79.3±10%. The data are consistent with thepresence of free Mab 166-32 in the circulation until 18 hours after CPB(FIG. 39). In hemolytic assays using sensitized chicken red blood cellsto measure the classical complement activity, the corresponding plasmasamples from the Mab 166-32 treated animals did not show any reductionin the classical complement activity by the antibody (FIG. 40). Theseresults confirm that Mab 166-32 is a specific inhibitor of thealternative complement pathway.

The specificity of Mab 166-32 in inhibiting the alternative complementpathway was also demonstrated by the complete inhibition of Bb formation(FIG. 41). Bb is the activation product of factor B upon proteolyticcleavage by factor D. The increase in Bb formation in the controlanimals is attributed to the activation of the alternative complementpathway during CPB. The reduction of plasma Bb concentration below thebaseline in the Mab 166-32 treated animals could be due to theinhibition of the physiological activation of the alternative complementin the animals.

Activation of the classical complement pathway was determined bymeasuring C4d, which is a specific marker for the activation of C4 inthe classical complement pathway. In the study, the plasma levels of C4din both the Mab 166-32 treated and control animals were relativelystable with reference to the baseline (FIG. 42). However, there was anincrease of C4d in both animal groups after neutralization of heparinwith protamine at the end of the CPB, indicating the activation of theclassical pathway as reported earlier in other studies (Kirklin J K etal., Ann. Thorac. Surg. 1986; 41: 193-199; Carr J A et al., J.Cardiovasc. Surg. (Torino) 1999; 40: 659-666).

Activation of complement is shown by an increase in plasma C3a in thecontrol animals (FIG. 43). In contrast, animals treated with Mab 166-32show almost complete inhibition of C3a production. A slight increase ofC3a level is observed in the Mab 166-32 treated animals afterneutralization of heparin with protamine. Together, the results fromFIGS. 41, 42 and 43 support the notion that complement activation duringCPB is predominantly via the alternative complement pathway. Due to thelack of cross-reactivity of the reagents in the commercial ELISA kitsfor baboon C5a and sC5b-9 (Quidel and Becton Dickinson, respectively),their concentrations were not determined.

Activation of neutrophils and monocytes in the baboons was examined bymeasuring CD11b (α-integrin) expression using immunofluorcytometricmethods. In the control animals, the CD11b expression on neutrophilsincreased rapidly and reached the maximum at about 85 minutes after thestart of CPB (209±42.9% of the baseline) (FIG. 44). It then declinedslowly back to around the baseline. In contrast, the increase of CD 11bexpression on neutrophils in the Mab 166-32 treated animals was delayedand smaller in magnitude (FIG. 44). The maximum level of CD11bexpression was 129.3%±5.5% of the baseline. Inhibition of the increasein CD11b expression on monocytes from the Mab 166-32 treated animals wasalso observed (FIG. 45).

Activation of platelets in the baboons was examined by measuring CD62P(P-selectin) expression using immunofluorocytometric methods. Both thecontrol and Mab 166-32 treated animals show a similar pattern ofinhibition of CD62P expression (FIG. 46). The exact cause of theinhibition of CD62P expression is unclear.

The effect of Mab 166-32 treatment on pro-inflammatory cytokines wasalso examined. Baboons treated with Mab 166-32 had a significantlysmaller increase in plasma IL-6 concentration as compared to the controlanimals (FIG. 47).

The effects of Mab 166-32 treatment on tissue injury of various organswere also studied. The increase of plasma LDH levels was significantlyreduced in the Mab 166-32 treated animals at 3 and 6 hours after CPB(FIG. 48). This indicates protection against tissue injury. Specificallyfor myocardial injury, the increase of both plasma CK and CK-MB levelsat 6 and 18 hours after CPB was significantly lower in the Mab 166-32treated animals as compared to the control animals (FIGS. 49 and 50).

As for pulmonary functions, the dynamic lung compliance of the Mab166-32 treated animals was higher than that of the control animalsduring the early phase of the open-chest surgery (FIG. 51). However,there was no significant difference in the lung compliance between thetwo groups of animals during and after CPB. The initial increase of thedynamic lung compliance in the control animals is probably attributed tothe open-chest procedure. The higher dynamic lung compliance in the Mab166-32 treated animals could be due to the protection against surgicaltrauma which was shown to be associated with complement activation viathe alternative complement pathway. (Gu Y J et al., Chest 1999; 116:892-898).

The renal function of the baboons was also examined. A reduction in theincrease of plasma creatinine was found in the baboons treated with Mab166-32 as compared to the control animals at 18 hours after CPB (FIG.52).

In conclusion, anti-factor D Mab 166-32 is effective in inhibiting theactivation of the alternative complement pathway in a baboon model ofCPB. Inhibition of the alternative complement pathway by Mab 166-32effectively reduces the activation of neutrophils and monocytes, as wellas the production of IL6. Treatment with Mab 166-32 confers protectionagainst myocardial and renal injury. The alternative complement pathwaymay play a predominant role in the inflammation and tissue injury causedby extracorporeal circulation, surgical trauma and ischemia/reperfusion.Therefore Mab 166-32 could be potentially useful for the treatment ofsystemic inflammatory response syndromes in CPB.

The foregoing description, terms, expressions and examples are exemplaryonly and not limiting. The invention includes all equivalents of theforegoing embodiments, both known and unknown. The invention is limitedonly by the claims which follow and not by any statement in any otherportion of this document or in any other source.

1-21. (canceled)
 22. A method of ameliorating a disease or conditionassociated with excessive or uncontrolled alternative pathway complementactivation comprising administering a sufficient amount of an antibodythat specifically binds to Factor D, or a Factor D binding fragmentthereof, to completely inhibit alternative pathway complement activationat a molar ratio of antibody to Factor D of about 1.5:1, wherein thedisease or condition is an inflammatory condition, an adverse drugreaction, or an autoimmune disease.
 23. The method of claim 22, whereinthe disease or condition is selected from the group consisting of severeburns, endotoxemia, septic shock, adult respiratory distress syndrome,hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn'sdisease, sickle cell anemia, poststreptococcal glomerulonephritis andpancreatitis.
 24. The method of claim 22, wherein the drug reaction isdue to a drug allergy, IL-2 induced vascular leakage syndrome orradiographic contrast media allergy.
 25. The method of claim 22, whereinthe autoimmune disease is selected from the group consisting of systemiclupus erythematosus, myasthenia gravis, rheumatoid arthritis,Alzheimer's disease and multiple sclerosis.
 26. The method of claim 22,wherein the antibody, or binding fragment thereof, binds to a region ofhuman factor D SEQ ID NO 2 from amino acid residue Cys154 to Cys170(inclusive).
 27. The method of claim 26, wherein said region comprisesamino acid residues Arg156, His 159, and Leu
 168. 28. The method ofclaim 22, wherein the antibody is monoclonal.
 29. The method of claim28, wherein the antibody is a chimeric, humanized, deimmunised or humanantibody.
 30. The method of claim 28, wherein the antibody is monoclonalantibody 166-32 produced by the hybridoma cell line deposited under ATCCAccession Number HB-12476.
 31. The method of claim 22, wherein theantibody comprises the murine variable regions of the antibodydesignated 166-32 produced by the hybridoma cell line deposited underATCC Accession Number HB-12476 and human constant regions.
 32. Themethod of claim 22, wherein the antibody, or binding fragment thereof,binds to the same epitope on human factor D as the antibody designated166-32 produced by the hybridoma cell line deposited under ATCCAccession Number HB-12476.